Guggulphospholipid methods and compositions

ABSTRACT

The present invention relates to the methods for preparing synthetic guggulphospholipids, their fatty acid analogues and other bioactive molecules. The present invention relates to E-guggulsterone and Z-guggulsterone or mixture of E- and Z-guggulsterones, and E-guggulsterol and Z-guggulsterol or mixture of E- and Z-guggulsterols synthetically modified to guggulphospholipids and analogues and salts thereof, fatty acid analogues of guggulsterols, guggulsulfate and salts thereof, guggulphosphate and salts thereof; and guggulsterols conjugated with drugs for use as prodrugs. Also the present invention provides a novel method for the preparation of E-guggulsterol and Z-guggulsterol or mixture of E- and Z-guggulsterols from a mixture of E- and Z-guggulsterones. The present invention further relates to guggulphospholipids and other bioactive molecules incorporated into complexes such as liposomes, complexes, emulsions, vesicles, micelles, and mixed micelles, which can include other active agents, such as hydrophobic or hydrophilic drugs for use, e.g., in treatment of human and animal diseases.

This application claims priority to U.S. Provisional Application SerialNo. 60/856,952, filed Nov. 06, 2007, which is incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to synthesis of novel phospholipid analogues andvariants of E- and Z-guggulsterones or gugguisterols or mixtures of E-and Z-guggulsterones or E- and Z-guggulsterols, and their targetdelivery for the treatment of human and animal diseases.

BACKGROUND OF THE INVENTION

Ayurveda practitioners take a holistic view of human disease. It viewsany disease as a dysfunction of the whole body rather than of a singleorgan or physiological process. Most of the Ayurvedic drugs thereforeare likely to act on a number of dysfunctions of the body involving anumber of organs and functions. In the 1960s the oleogum resin (gumguggul), isolated from a small tree Commiphora wighitii belonging to thefamily Burseraceae, was studied for its potential use in the treatmentof elevated blood cholesterol or hyperlipidemia. The Commiphora tree, ingeneral, is mentioned in the classic Ayurvedic literature for use in thetreatment of bone fractures, arthritis, obesity, inflammation,cardiovascular diseases and lipid disorders. With the discovery of thehypolipidemic activity for the gum guggul, several chemicalinvestigations were undertaken and found that gum guggul is a complexmixture of various classes of chemical compounds such as linans, lipids,diterpenoids, and plant steroids. Based on the structure functionanalysis of gum guggul, it was determined that the soluble portion ofthe gum in ethyl acetate, and specifically its neutral portion,contained most of the hypolipidemic properties. The neutral fraction wasfound to be source of sterol compounds known as Z- and E-guggulsterone,present in an amount minimum of 2.5% and responsible for lowering ofblood cholesterol [Indian Pharmacopea, 1988 G. V. Satyavati, Economicand Medical Plant Research. 5, 47 (1991)].

Modern pharmacological studies on the crude drug and some of itsfraction have supported the claims of Ayurveda. The anti-arthritis andanti-inflammatory activities were confirmed by Gujral et al. [M. L.Gujral, K. Sareen, K. K. Tangri, Amma, and A. K. Roy, Ind. J Physiol.Pharmacol. 4, 267 (1960)]. A mixed type of mechanism has been implicatedfor lipid lowering effect of gum guggul. The stimulation of plasma LCAT,hepatic lipases, receptor mediated catabolism of LDL and increasedfaecal bile acid excretion as well as suppression of hepatic cholesterolbiosynthesis are the mechanisms responsible for lipid lowering effect ofgum guggul [S. Nityanand and N. K. Kapoor, Ind J Exp. Biol. 11, 395(1973); N. K. Kapoor and S. Nityanand, Ind. J Heart Res. Supp—1.22(1988)]. With the discovery of the hypolipidemic activity for the gumresin, systemic chemical investigations were carried out to characterizecompounds of the gum resin responsible for hypolipidemic activity. McCook et al. have claimed alcoholic extract of gum guggul for controllingor preventing sebum secretion from sebocytes which is associated with ashiny, undesirable appearance and a disagreeable tactile sensation [J.P. Mc Cook et al. U.S. Pat. No.5,690,948 (1997)].

As is already known, cholesterol is the precursor of neurosteroidpregnenlone. Similarity among biogenic precursor of pregnenolone andguggulsterones or guggulsterol [V. D. Patil, U. R. Nayak, and Sukh Dev,Tetrahedron 28, 2341 (1972)] has prompted exploration of the propertiesof synthetically pure pharmaceutically active novel phospholipid bondedto guggulsterone or guggulsterol.

Behavioral studies have suggested a potential role of pregnenolone formemory enhancement. Intracerebroventricular (i.c.v) administration ofpregnenolone and pregnenolone sulfate leads to amelioration in variousmemory tasks in rodents [IF. Flood. J. F. Moorley, and E. Robert. Proc.Natl. Acad Sci. USA 89, 1567 (1992)]. These memory-enhancing effectsmight be attributed to the N-methyl-D-aspartate (NMDA)-antagonisticproperties of pregnenolone sulfate since NMDA agonists have been shownto impair cognitive functions in rodents [M. R. Bowlby, Mol. Pharmacol.43,813 (1993)].

In recent years, interest is increasing to understand the role of freeradical oxidative damage in human diseases. Free radicals are highlyreactive species that have the potential to oxidize biological membranesincluding proteins, lipids and. DNA. To prevent or reduce oxidation,rich arrays of natural antioxidant mechanism exist. These antioxidantdefense mechanisms have been found defective in many diseases. Increasedproduction of free radicals has been strongly implicated in thepathophysiology of diabetes and atherosclerosis. Glucose combines withserum proteins and lipoproteins in a non-enzymatic glycation reactionand may auto oxidize in situ, generating free radicals and causing localoxidative damage [J. V. Hunt and S. P. Wolff, Free Radical Res. Commun.12-13, 115 (1991)]. The free radical scavenging antioxidants reactpreferentially with free radicals before vital structure can beattacked. The gum guggul and E- and Z-guggulsterones are also known asto have antioxidant property [K. Singh, R. Chander, and N. K. Kapoor,Phytotherapy Research, 11, 291 (1997)].

The guggulphospholipid methods and compositions of the present inventionfind application in each of these areas.

SUMMARY OF THE INVENTION

The present invention relates to novel guggulphospholipid compositionsand methods of synthesis and uses thereof In some embodiments, theinvention pertains to synthetic, pharmaceutically active phospholipidsconjugated with E-guggulsterone and Z-guggulsterone or guggulsterols, ormixture of E-guggulsterone and Z-guggulsterone or mixture ofE-guggulsterol and Z-guggulsterol and their analogues. Phospholipids aregenerally used for drug delivery. The guggulphospholipids and otherbioactive products of the present invention help to target delivery ofnovel products, with or without additional therapeutically activeproducts, for the treatment of human or animal diseases, such asprevention and treatment of abnormal cell growth, cancer, cardiovasculardisease, viral infection, skin infections, memory loss, obesity,proliferation in inflammation, etc.

The compositions of the present invention find use, for example, in theprevention and treatment of abnormal cell growth, proliferation ininflammation, lowering the elevated low density lipoprotein (LDL) andhigh levels of cholesterol, and elevating the low levels of the highdensity lipoprotein (HDL), neoplastic and cardiovascular diseases inhumans and animals. This invention also provides methods of using thedisclosed compositions for preventing or controlling cognitivedysfunction, hyperglycemia and skin infection.

In some embodiments, the present invention comprises guggul derivativeshaving general structures III-XII, as diagrammed in FIG. 1, In someembodiments, of structures III and IV, R₁ is a saturated or unsaturatedacyl or alkyl groups having between 1 and 34 carbon atoms. In someembodiments, the guggul derivative comprises structure IV, and R₁ is asugar. In some embodiments, the guggul derivative comprises structure V,wherein R₁ and R₂ are same or different and at least one of the R₁ or R₂is a saturated or unsaturated acyl group or alkyl group having between 1and 34 carbon atoms.

In some embodiments of guggul derivatives comprising structures IV-X,side group X is hydrogen, methyl, ammonium, sodium, potassium, calcium,barium ion or any non-toxic ion.

In some embodiments of a guggul derivative comprising structures VI, Xand Y are the same or different, and are hydrogen, methyl, hydrogen,ammonium, sodium, potassium, calcium, barium ion or any non-toxic ion.

In some embodiments of a guggul derivative comprising structure VIII R₃,R₄ and R₅ are the same or different, and are hydrogen or methyl group.Formula VIII is a cationic guggul derivative when X is a methyl group.

In some embodiments of a guggul derivative comprising structure X, PEG(polyethylene glycol) is a long chain, linear or branched syntheticpolymer composed of ethylene oxide units, HO(CH₂CH₂O)_(n)CH₂CH₂OCH₃,where n is typically between about 1 and about 1000.

In some embodiments of guggul derivative comprising structure XI, thedrug is an active agent.

Formula XII is a cationic guggul derivative, wherein R₆, R₇, and R₈ arethe same or different and are hydrogen, methyl, alkyl, substitutedalkyl, alkyoxy, substituted alkyloxy groups, are optionallyhydroxylated, aminated, and/or polyaminated, the guggul derivativehaving overall positive charge.

In some embodiments, guggul derivatives comprising structures HI-XII arein a form selected from the group consisting of E-isomers, Z-isomers, ora mixture of E and Z isomers.

In some embodiments, guggul derivatives having structures III-XII areoptically pure, while in some embodiments, the structures III-XII aremixtures of optical isomers.

In some embodiments, the present invention provides methods of preparingguggul derivatives of formula from guggulsterol having structure IIA.

In some embodiments, the guggulsterol IIA is an E-isomer, while in otherembodiments, it is a Z-isomer, while in still other embodiments, it is amixture of E and Z isomers. In some embodiments the guggulsterol isoptically pure, while in some embodiments, the guggulsterol is a mixtureof optical isomers. The present invention also provides methods ofpreparing guggulsterol IIA from guggulsterone I.

In some embodiments, the present invention provides methods of preparingguggul derivatives of formula III-XII from guggulsterone havingstructure I (shown in FIG. 2). In some embodiments, the guggulsterone Iis an E-isomer, while in other embodiments, it is a Z-isomer, while instill other embodiments, it is a mixture of E and Z isomers.

In some embodiments of the methods of synthesizing a guggul derivativefrom a guggulsterol IIA or a guggulsterone I, the guggul derivatives(III-XII) are synthesized from the precursor in a single step, while insome embodiments, the guggul derivatives are synthesized in a sequenceof multiple steps.

In some embodiments, the guggulsterol is prepared in a single step or asequence of many steps, while in some embodiments, the guggulsterol isobtained, e.g., commercially.

In some embodiments of the methods of preparing compounds of formulasIV-VI, VIII-X, at least one step comprises the use of phosphoramiditereagent or a phosphorylating agent. In some preferred embodiments, thephosphoramidite reagent includes but not limited toN,N-diisopropylmethylphosphoramidic chloride,(benzyloxy)(N,N-diisopropylamino)chlorophosphine, benzyloxybis(diisopropylamino) phosphine,2-cyanoethyl-N,N,N,N-tetraisopropylphosphoramidite,(2-cyanoethyl)(N,N-diisopropylamino)chlorophosphine, difluorenyldiisopropylphosphoramidite, methyl-N,N,N,Ntetraisopropylphosphorodiamidite, dimethylN,N-diisopropylphosphoramidite, dibenzyl diisopropylphosphoramidite,di-text-butyl-N,N-diisopropylphosphoramidite,2-(diphenylmethylsilyl)ethyl-N,N,N,N-tetraisopropylphosphoramidite,(N-trifluoroacetylamino) butyl and (N-trifluoroacetylamino)pentyl-N,N,N,N-tetraisopropylphosphoramidites. In some embodiments, thephosphorylating agent includes but is not limited to2-bromoethyldichlorophosphate, trimethylsilylethyl dichlorophosphate,methyl dichlorophosphate, 2-chloro-2-oxo-1,3,2-dioxaphospholane,2-chlorophenyl dichlorophosphate, and phosphorus oxychloride.

In some embodiments of the methods of the present invention, structureXI is prepared by conjugating guggulsterol with a drug, e.g., directlyor through a linker. In some embodiments in which a linker is used, thelinker is an alkyl group, which is optionally substituted withfunctional groups such as carbonyl, carboxyl, carbonate, amino, amide,ester, thioester groups, succinate, glutarate, carbamate, ether;phosphate; phosphonate, diphosphate; pyrophosphate; and the like.

In some embodiments, the methods of the present invention compriseformation of complexes including but not limited to liposomes, whereinthe liposomes or complexes comprise guggul derivatives III-XII. In somepreferred embodiments, the complexes are micelles, vesicles, oremulsions. In some embodiments, the methods of the present inventioncomprise formation of a composition comprising a plurality of micelles,wherein the micelles are in the form of monomeric, dimeric, polymeric ormixed micelles.

In some embodiments, the present invention comprises a method ofretaining drug in liposomes or complexes, comprising preparing guggulderivatives by the methods described herein, and including the guggulderivatives and a drug in a liposome or complex, In some embodiments,the present invention comprises a liposomal composition prepared asdescribed above,

In some embodiments, the present invention comprises a method ofcomplexing a drug with a guggul derivative III-XII. In sonicembodiments, the guggul derivative is in a complex prior to complexingwith the drug.

In some embodiments, the invention comprises any of the methodsdescribed above, wherein the complexes includes, micelles, mixedmicelles, polymeric micelles, vesicles, emulsions and liposomes.

In some embodiments of the present invention, the compositions of themethods described above further comprise phospholipids, pegylatedphospholipids, polyethylene glycol (PEG), fatty acids, sterols, cholicacid, or a tocopherol. In some embodiments, the cholic acid is sodiumdeoxycholate. In some preferred embodiments comprising the use of PEG,the PEG has an average molecular weight of 200-20,000. In embodimentscomprising fatty acids, in some embodiments, at least one of the fattyacids is selected from a group consisting of fatty acids having chainlength of C₄-C₃₄. In some embodiments, at least one of the fatty acidsis saturated, while in some embodiments, at least one of the fatty acidsis unsaturated.

In some embodiments, at least one fatty acid is in acidic form, while inother embodiments; at least one fatty acid is in salt form.

In certain preferred embodiments, at least one of the phospholipidscomprises a phosphatidylcholine, a phosphatidylethanolamine, aphosphatidylglycerol, a phosphatidylserine, a phosphatidylinositol, aphosphatidic acid. In particularly preferred embodiments, at least oneof said phosphatidylcholine is dimyristoylphosphatidylcholine,disteroylphosphatidylcholine, dipalmitoylphosphatidylcholine,dioleovlphosphatidycholine, diarachidonoylphosphatidylcholine, eggphosphatidylcholine, soy phosphatidylcholine, hydrogenated soyphosphatidylcholine, partially hydrogenated soy phosphatidylcholine, ora mixture thereof. In additional preferred embodiments, at least one ofsaid phosphatidylethanolamine is dimyristoylphosphatidylethanolamine,disteroylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine,dioleoylphosphatidyethanolamine, diarachidonoylphosphatidylethanolamine,egg phosphatidylethanolamine, or a mixture thereof.

In some embodiments, at least one of said phosphatidylglycerol isdimyristoylphosphatidylalycerol, disteroylphosphatidylglycerol,dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol,diarachidonoylphosphatidylglycerol, or a mixture thereof.

In some embodiments, at least one of said phosphatidylserine isdimyristoylphosphatidylserine, disteroylphosphatidylserine,dipahnitoylphosphatidylserine, dioleoylphosphatidylserine,diarachidonoylphosphatidylserine, or a mixture thereof.

In some embodiments, at least one of said phosphatidylinositol isdimyristoylphosphatidylinositol, disteroylphosphatidylinositol,dipalmitoylphosphatidyinositol, dioleoylphosphatidylinositol,diarachidonoylphosphatidylinositol, or a mixture thereof.

In some embodiments, at least one of said phosphatidic acid isdimyristoylphosphatidic acid, disteroylphosphatidic acid,dipalmitoylphosphatidic acid, dioleoylphosphatidic acid,diarachidonoylphosphatidic acid or a mixture thereof.

In some embodiments, at least one of said pegylated phospholipidscomprises a pegylated derivative of phospholipids selected from thegroup consisting of pegylated derivatives ofdisteroylhosphatidylglycerol, dimyristoylhosphatidylglycerol, anddioleoylphosphtidylglycerol.

In some embodiments, the structure XI is prepared by conjugating aguggulsterol with a drug directly or through a linker, and thecomposition further comprises a sterol selected from a group consistingof cholesterol, derivatives of cholesterol, cholesteryl sulfate,cholesterol succinate, cholesterol hemisuccinate, cholesterol oleate,cortisol, corticosterone, hydrocortisone, cholesterol-PEG, coprostanol,cholestanol, cholestane, β-sitosterol, lanosterol, campesterol,lathosterol, stigmasterol, stigmastanol, calciferol, or a mixturethereof

In some embodiments of the methods and compositions of the presentinvention, the composition comprises at least one drug and the at leastone drug is a therapeutically active agent. In some preferredembodiments, at least one therapeutically active agent comprises ananticancer drug, while in some preferred embodiments; at least onetherapeutically active agent comprises an antiviral drug. In yet morepreferred embodiments, at least one therapeutically active agentcomprises an antifungal drug.

In preferred embodiments of the methods and compositions of the presentinvention, at least one of said therapeutically active agents is totreat a condition selected from the group consisting of cardiovasculardisease, neoplasia, memory loss, inflammation, anti-inflammation drug,skin infection or skin disease, serum cholesterol level, andhyperglycemia. In some preferred embodiments, at least one of saidtherapeutically active agents improves cognitive function.

In some embodiments of the methods and compositions of the presentinvention, the composition comprises a liposome composition comprisingunilamellar vesicles, multilamellar vesicles, or mixtures thereof. Insome embodiments, the liposomes or any of the complexes described hereinhave an overall negative charge, while in some embodiments they have anoverall positive charge. In some embodiments, they have an overallneutral charge.

In some embodiments of the methods and compositions of the presentinvention, the composition comprises a liposome or complex composition,wherein the liposome or complex has a mean diameter of about 20 micronor less. In some preferred embodiments, the liposome or complex has amean diameter of about 10 micron or less, while in some preferredembodiments, the liposome or complex has a mean diameter of about 5micron or less. In some particularly preferred embodiments, the liposomeor complex has a mean diameter of about 1 micron or

In some embodiments of the methods and compositions of the presentinvention, the composition is in lyophilized form. In some embodiments,the lyophilized composition further comprises a cryoprotectant such asone or more sugars, In preferred embodiments, the cryoprotectant is asugar selected from a group consisting of trehalose, maltose, lactose,sucrose, and dextran.

In some embodiments of the methods and compositions of the presentinvention, the liposome or complex composition comprising one or moreguggul derivatives is in form selected from the group consisting of apowder form, a solution form, a suspension form, an emulsion form, amicelle form, a gel form, or a paste form.

In some embodiments, a liposome or complex composition according to thepresent invention is encapsulated in a capsule. In preferredembodiments, the capsule comprises an enteric coating. “Enteric” refersto the small intestine, therefore “enteric coating” generally refers toa coating that substantially prevents release of a medication before itreaches the small intestine. While not limiting the invention to anyparticular mechanism of action, it is understood that most entericcoatings work by presenting a surface that is stable at acidic pH butbreaks down rapidly at higher pH.

In some embodiments, the present inventions comprise a method oftreating a cell with a composition comprising a guggul derivativecomposition as described herein, comprising preparing a composition asdescribed herein, and exposing the cell to the composition, in somepreferred embodiments, the exposing of the cell occurs in vivo, e.g., ina patient or subject.

It is contemplated that in some embodiments, the exposing of a cell in asubject comprises oral delivery of the composition to the subject, whilein other embodiments; the exposing of a cell comprises intravenousdelivery of the composition to the subject. Routes of delivery of thecomposition to the subject that find use in the present inventioninclude but are not limited to subcutaneous delivery, parenteraldelivery, intraperitoneal delivery, rectal delivery, vaginal deliveryand/or topical delivery. In some preferred embodiments, the subject is amammal. In some particularly preferred embodiments, the mammal is human.

In some embodiments, the present invention comprises a method oftreating a human or animal disease, comprising administering atherapeutically effective amount of a composition comprising a guggulderivative as described herein, and exposing the composition to a humanor animal in need thereof, such that the composition is delivered to thehuman or animal patient.

In some embodiments, the present invention comprises a method oftreating a human or animal disease, comprising administering atherapeutically effective amount of a composition comprising a liposomeor complex comprising a guggul derivative as described herein, andexposing the composition to a human or animal in need thereof such thatthe active agent is delivered to the human or animal patient.

In some embodiments, the present invention comprises a method ofalleviation of a human or animal disease, comprising administering atherapeutically effective amount of a composition comprising a guggulderivative as described herein, and exposing the composition to a humanor animal in need thereof such that the composition is delivered to thehuman or animal patient.

In some embodiments, the present invention comprises a method ofalleviation of a human or animal disease, comprising preparing aliposome or complex comprising a guggul derivative as described herein,and exposing the composition to a human or animal in need thereof suchthat the composition is delivered to the human or animal patient.

In some embodiments the treated animal is a mammal. In certainembodiments, the disease is a cancer disease. In certain preferredembodiments, cancer disease is selected from a group consisting ofcancers of head, neck, brain, blood, bone, breast, lung, pancreas,spleen, bladder, prostate, testes, colon, kidney, uterus, ovary, skin,bone marrow, esophagus, stomach, intestine, larynx, tongue, and mouth.

In some embodiments, the disease is a viral disease. In certainpreferred embodiments the viral disease is selected from a groupconsisting of HIV, herpes simplex viruses, human herpes virus 6, humanherpes virus 7, human herpes virus 8, orthopoxviruses, ebola virus,influenza virus, tuberculosis, hepatitis A, hepatitis B, hepatitis C,hepatitis D, hepatitis E, hepatitis G, parainfluenza virus, respiratorysyncytial virus, cholera, pneumonia, SARS virus, canary virus, West Nilevirus (WNV), respiratory syncytial virus (RSV), dengue virus, varicellazoster virus, corona viruses, vaccinia virus, cytomegalovirus (CMV),human rhinovirus (HRV), papilloma virus (PV) and Epstein Barr viruses.

In some embodiments, the methods and compositions of the presentinvention are used in the treatment of a disease selected from the groupconsisting of fungal disease, cardiovascular disease, neoplasia,Alzheimer disease, an inflammatory disease, skin disease, hyperlipidemiadisease, and a cognitive dysfunction disease.

Particular embodiments of the invention are described in this Summary,and below, in the Brief and Detailed descriptions of the invention.Although the invention has been described in connection with specificembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrams compound IIA and compounds III-XII

FIG. 2 diagrams compound I and compounds IIA-IIF

FIG. 3 diagrams an exemplary synthetic scheme for compound IA.

FIG. 4 diagrams an exemplary synthetic scheme for compound VII.

FIG. 5 diagrams an exemplary synthetic scheme for compound III.

FIG. 6 diagrams an exemplary synthetic scheme for compound VI.

FIG. 7 diagrams an exemplary synthetic scheme for compound V.

FIG. 8 diagrams an exemplary synthetic scheme for compound V.

FIG. 9 diagrams an exemplary synthetic scheme for compound VIII.

DEFINITIONS

As used herein, the term “effective amount” refers to the amount of anactive composition (e.g., a pharmaceutical compound or compositionprovided as a component in a lipid formulation) sufficient to effectbeneficial or desired results. An effective amount can be administeredin one or more administrations, applications or dosages and is notintended to be limited to a particular formulation or administrationroute.

As used herein, the terms “active” or “pharmaceutically active” as usedin reference to an agent, composition, or compound, refers to an agentthat, upon administration or application, causes a beneficial, desired,or expected result. The administration may be in one or moreadministrations, applications or dosages and is not intended to belimited to a particular formulation or administration route. The term isnot limited to any particular level of activity.

The terms “agent” and “compound” are used herein interchangeably torefer to any atom, molecule, mixture, or more complex composition havingan attributed feature. For example, an “active agent” or “activecompound” refers to any atom, molecule, preparation, mixture, etc.,that, upon administration or application, causes a beneficial, desired,or expected result.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, or other active agent, or therapeutic treatment (e.g.,compositions of the present invention) to a physiological system (e.g.,a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs).Exemplary routes of administration to the human body can be through theeyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs(inhalant), rectal, vaginal, oral mucosa (buccal), ear, by injection(e.g., intravenously, subcutaneously, intratumorally, intraperitoneally,etc.) and the like. Administration may be in one or moreadministrations, applications or dosages, and is not intended to belimited to a particular administration route.

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) (e.g., two separate lipidcompositions, containing different active compounds) or therapies to asubject. In some embodiments, the co-administration of two or moreagents or therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents or therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents or therapiesare co-administered, the respective agents or therapies are administeredat lower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of the agents or therapies lowers the requisite dosageof a potentially harmful (e.g., toxic) agent(s).

As used herein, the term “toxic” refers to any detrimental or harmfuleffects on a subject, a cell, or a tissue as compared to the same cellor tissue prior to the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent (e.g., an active pharmaceutical compound)with a carrier, inert or active (e.g., a phospholipid), making thecomposition especially suitable for diagnostic or therapeutic use invitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “topically” refers to application of thecompositions of the present invention to the surface of the skin andmucosa! cells and tissues (e.g., alveolar, buccal, lingual, masticatory,or nasal mucosa, and other tissues and cells which line hollow organs orbody cavities).

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintrigrants (e.g.,potato starch or sodium starch glycolate), and the like., Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers, and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975), incorporated herein by reference). Moreover, in certainembodiments, the compositions of the present invention may be formulatedfor horticultural or agricultural use. Such formulations include dips,sprays, seed dressings, stem injections, sprays, and mists.

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound of the present invention that is physiologically tolerated inthe target subject (e.g., a mammalian subject, and/or in vivo or exvivo, cells, tissues, or organs). “Salts” of the compounds of thepresent invention may be derived from inorganic or organic acids andbases, Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like,

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, palmate, pectinate,persulfate, phenylpropionate, picrate, pivalate, propionate, succinate,tartrate, thiocyanate, tosylate, undecanoate, and the like. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like. For therapeutic use,salts of the compounds of the present invention are contemplated asbeing pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

The term “Polyethylene glycol (PEG)” includes polymers of lower alkyleneoxide, in particular ethylene oxide (polyethylene glycols) having anesterifiable hydroxyl group at least at one end of the polymer molecule,as well as derivatives of such polymers having esterifiable carboxygroups. Polyethylene glycols of an average molecular weight ranging from200-20,000 are preferred; those having an average molecular weightranging from 500-2000 are particularly preferred.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions, and usesthereof, for the treatment of mammalian diseases. More particularly, thepresent invention relates to synthesis and uses of thesepharmaceutically active phospholipids, their analogues and variants asdescribed above and compositions that contain them, generally to thetreatment of cholesterol lowering and weight loss, antileukemic effects,skin diseases, viral infections, as memory enhancing drug, and cancerprevention or treatment in general. The pharmaceutically activephospholipids prepared by the present method can be incorporated intoliposomes, and other formulations like complexes, emulsions, vesicles,miscalls, which can also include other active agents such as hydrophobicor hydrophilic drugs. Such liposomes or lipid formulations can be usedto treat human or animal diseases. Liposomes can also include ligandsfor targeting a particular cell type or specific tissue.

A disease may be a neoplasia, a neurodegenerative, a pulmonary disease,obesity, an inflammatory disease and a cardiovascular disease. Morepreferably, a neoplasia is a solid tumor, leukemia or lymphoma. Morepreferably, a neurodegenerative disease is Alzheimer's disease. Theconcept of Alzheimer's disease as an inflammatory disease has increasedinterest in the use of anti-inflammatory agents in improving memoryloss. The present invention provides a series of novel compounds thatcan be useful for the treatment of neurodegenerative and other diseasesmentioned above.

Animal studies have shown a positive correlation between dietary fat andthe rate of tumor growth and the severity of metastases. In humans thelink between nutrition and cancer is speculation, in particular theassociation between dietary fat and the origins of human colorectal,breast, prostatic, ovarian and endometrial cancers, It is recognizedthat the rate of lipid synthesis in tumor cells is quite rapid. Thisphenomenon can be understood, because rapidly dividing cells not onlyneed fresh copies of DNA and proteins, but also require the security ofnew biomembrane composed of phospholipids and cholesterol. Thecholesterol synthesizing pathway has been implicated as a promoter oftumor cell growth. The other major classes of lipids, fatty acids, arealso involved in tumor cell growth. In fact many human and experimentalcancers express elevated levels of fatty acid synthase (FAS), the enzymerequired for endogenous fatty acid biosynthesis. As for example, aprognostic molecule isolated from a number of breast cancer patients wasidentified as FAS. Inhibition of FAS leads to loss of clonogeniccapacity and induction of programmed cell death in Breast cancer cells.The structure of guggulsterones or gugguisterol of naturally occurringguggul resin is very similar to cholesterol. It is proposed thatguggulphospholipids of the invention will mimic the cancer cells assource of phospholipids, fatty acids and cholesterol. It is proposedthat the potential anti-cancer treatment includes: (a) blocking de novosynthesis of all cholesterol intermediates leading to an inability ofRas to stimulate cell growth and division, and (b) reducing theprecursor supply for fatty acid synthase (FAS) and thus blocking tumorcell proliferation.

The present invention provides liposome delivery systems for thecompositions of the present invention. Liposome formulations have thecapacity to increase the solubility of hydrophobic drugs in aqueoussolution. They often reduce the side effects associated with drugtherapy. Liposomes are commonly prepared from naturally occurringphospholipids such as phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol.Anionic phospholipids, such as phosphatidylglycerol, can be added togenerate negative surface charge that provides for colloidstabilization. These phospholipids are often purified from naturalsources, such as soybean and egg yolk, and also can be synthesizedchemically with same or different fatty acids (saturated orunsaturated).

The nature and density of the surface charge of liposomes influence onstability, kinetics, biodistribution, and interaction with, and uptakeby target cells. Liposome surface charge also influences the tendencyfor liposomes to aggregate, which makes liposome difficult to work withand affects uptake by target cells. Therefore, liposomes with a neutralsurface charge have the highest tendency to aggregate, but are lesslikely to be cleared by cells of the reticuloendothelial system (RES)after systemic administration. On the other hand, negatively chargedliposome exhibit reduced aggregation and increased stability and,therefore, exhibit non-specific cellular uptake in vivo. Thus, it hasbeen suggested that a small amount of negatively charged lipids maystabilize neutral liposome against an aggregation-dependent uptakemechanism [Drummond et al., Pharm. Rev. 51,691-743 (1999); D. C.Drummond, O. Meyer, K. Hong, D. B. Kirpotin, D. Papahadgopoulos, Pharm.Rev. 51,691-743 (1999).] The negatively charged lipids may be natural orsynthetically modified phospholipids. The present invention providessuch methods and compositions for improved liposome formulation.

The present invention relates to the use of more definedpharmaceutically active guggulphospholipids and other bioactivemolecules of present invention for treatment or prevention of abnormalcell growth, proliferation in inflammation, cholesterol lowering andweight loss, skin diseases such as acne affections, memory loss, andAlzheimer disease, neoplastic and cardiovascular diseases. Anotherobject of the invention is the use of possible cationic or anionic orPEGylated analogues of pharmaceutically active novel compounds ofpresent invention as drug carrier. Another object of this invention isthe use of fatty acid-conjugated guggulsterones or guggulsterol in acomposition with or without active agents or drugs and their uses in thetreatment of human and animal diseases. Fatty acids may be short chainor long chain, saturated or unsaturated [e.g., guggulmyristate (C14:0);guggullinoleate (C18:3), etc.]. Additionally, the methods of theinvention comprise administering a compound of invention as drug or in apharmaceutical composition to combat mammalian diseases.

Synthetic guggulphospholipids of the present invention can convenientlybe incorporated into liposomes, emulsion, micelles, vesicles, orcomplexes. Such liposome can also include one or more therapeutic agent.Such liposome and other formulations can be used to treat or alleviatecancer and other diseases, given orally in the form of liquid doses orinside enteric coated capsule or by i.v. injection. The formulation canbe applied as liposome lip gels, ointment or cream for the treatment ofskin diseases.

Another object of the present invention is to develop cognitionenhancing effect of pharmaceutically active guggulphospholipids andother bioactive molecules of present invention given orally, topically,parenterally or by other method of administration in any pharmaceuticalpreparations and with amount necessary for activity.

Another object of the present invention is to develop a method ofreducing, preventing or controlling hyperglycemic conditions byconsuming pharmaceutically active guggulphospholipids and otherbioactive products of the present invention in any pharmaceuticallyacceptable formulations. In addition, the guggulphospholipids can beconsumed as a nutritional supplement for weight control, fat loss andhyperlipidemic properties. In any pharmaceutically acceptableformulations, the products of invention can be used with or withoutother excipients, as for example, high fiber, phosphate salts selectedfrom the group consisting of calcium phosphate, potassium phosphate,sodium phosphate and the like, phospholipids (natural or synthetic)known in the art of liposome formulation. The phosphatidylcholine,preferably soy phosphatidylcholine or hydrogenated soyphosphatidylcholine will be used. Other phospholipids such asphosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidylglycerol, may also be used separately or mixture thereof.The phosphate salts are preferably a mixture of calcium, potassium andsodium salt. The weight control product may be administered in the formof capsule preferably enteric coated. capsule, tablet, soft gel capsule,emulsion, syrup, food bar and the like.

In another embodiment of the invention, the pharmaceutically activeproducts of the present invention is to use for the treatment ofpatients suffering from human memory dysfunction like Alzheimer'sdisease and Korasakoff's disease alone or in combination with othertreatments. The compounds of the invention provide a new strategy in thetreatment of neurodegenerative diseases, preferably Alzheimer's disease.

Another object of the present invention is to develop a method ofimproving conditions for the treatment of infected skin, for examplesome of the common fungal skin condition, and in general dermaldysfunctions.

Another object of the present invention is to develop a method ofreducing, preventing or controlling HIV, abnormal cell growth,proliferation in inflammation, neoplasia and cardiovascular diseases byconsuming novel products of the present invention in anypharmaceutically acceptable formulations,

One of the important objectives of the present invention is cancerprevention and treatment. Many animal studies have shown a definitepositive correlation between dietary fat and the rate of tumor growthand the severity of metastases. It is well known that lipid synthesis intumor cells is quite rapid. This phenomenon can be understood becauserapidly dividing cells not only need fresh copies of DNA and proteins,but they also require the security of new biomembranes composed ofphospholipids and cholesterol. The other major class of lipids, fattyacids, is also involved in tumor cell growth. Many human andexperimental cancers express elevated levels of fatty acid synthase(FAS), the major enzyme required for endogenous fatty acid biosynthesis.The novel guggulphospholipid may induce programmed cell death in cancercells.

An embodiment of the invention is the synthesis of compounds of presentinvention and their uses in liposomes, emulsion, micelles, vesicles andcomplexes. In the present invention, “biological activity” refers tocytotoxic, anti-proliferative, and antioxidant activity against a cell,more preferably a cell associated with a disease condition. Preferably,the cell exhibits abnormal cell growth. A “disease” is referred to aneoplasia, a neurodegenerative disease, a pulmonary disease, aninflammatory disease, a cardiovascular disease, or skin disease. Morepreferably, a neoplasia is a solid tumor, leukemia or lymphoma. Morepreferably, a neurodegenerative disease is Alzheimer's disease. Theindividual compound or mixture of compounds of the present invention canbe formulated into a pharmaceutical composition.

The invention may also regulate the synthesis of the vascularendothelial growth factor (VEGF). An embodiment of the invention is thestimulation of VEGF expression by the bioactive molecules of the presentinvention.

All products (e.g., compositions, complexes, etc.) of the presentinvention can be incorporated in common pharmaceutical compositions,such as are well known in the art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new and novel synthetic methods forproducing and using pharmaceutically active guggulphospholipids, theirfatty acid analogues and other bioactive molecules having the generalformulas I, IIA, III-XI (FIGS. 1, The compositions are useful for theprevention or treatment of conditions comprising but not limited toabnormal cell growth, proliferation in inflammation, cholesterollowering and weight loss, skin diseases, memory loss, Alzheimer disease,neoplastic and cardiovascular diseases in humans and animals. Thisinvention also provides their uses for preventing or controllingcognitive dysfunction, hyperglycemia, viral and skin infections. Thepresent invention describes to the synthetic methods and uses of thesepharmaceutically active molecules and compositions that contain them.

In Formulas III and IV, R₁ is a saturated or unsaturated acyl or alkylgroups having between 1 and 34 carbon atoms. In Formula V, at least oneof R₁ or R₂ is preferably a saturated or unsaturated acyl group or alkylgroup having between 1 and 34 carbon atoms. In a preferred embodiment,in Formulas V, R₁ and R₂ are the same and include from C₁ to C₃₄saturated and/or unsaturated acyl or alkyl group, preferably between 6and 24 carbon atoms and more preferably between 12 and 24 carbon atoms.

The term “alkyl” encompasses saturated or unsaturated straight chain andbranched-chain hydrocarbon moieties. The term “substituted alkyl”comprises alkyl groups further bearing one or more substituents selectedfrom hydroxyl, alkoxy (of a lower alkyl group), mercapto (of a loweralkyl group), cycloalkyl, substituted cycloalkyl, halogen, cyano, nitro,amino, amido, imino, thio, —C(O)H, acyl, oxyacyl (of a lower acyl)carboxyl and the like.

The term “acyl” encompasses saturated or unsaturated straight chain orbranched fatty acid chain. The term “substituted acyl” comprises acylgroups further bearing one or more substituents selected from hydroxyl,alkoxy (of a lower alkyl group), mercapto (of a lower alkyl group),cycloalkyl, substituted cycloalkyl, halogen, cyano, nitro, amino, amido,imino, thio, —C(O)H, acyl, oxyacyl (of a lower acyl) carboxyl and thelike. In formulas IV-X, X is hydrogen, methyl, ammonium, sodium,potassium, calcium, barium ion or any non-toxic ion, more preferablyhydrogen, sodium or ammonium ion. In Formula VI, X and Y are same ordifferent and are hydrogen, methyl, ammonium, sodium, potassium,calcium, barium ion or any non-toxic ion, more preferably hydrogen,sodium or ammonium ion.

In Formula IV, R₁ can also be sugar. Sugar is selected from a groupconsist of but not limited to allose, altrose, glucose, fructose,mannose, sucrose, galactose, sorbitol, glucosamine, glucuronic acid,talose, idose, gulose, ribose, deoxyribose, arabinose, xylose, and thelike.

In Formula VIII, R₃, R₄ and R₅ are same or different and are H or methylgroups. Formula VIII is cationic guggul derivative, wherein X is amethyl group.

The PEG (polyethylene glycol) group in Formula X is a long chain, linearor branched synthetic polymer composed of ethylene oxide units,HO(CH₂CH₂O)_(n),CH₂CH₂OCH₃, in which n is typically between about 1 andabout 1000 (such as between 1 and about 500) or otherwise can vary toprovide compounds with molecular weights (M.W.) from 200-50,000 Daltons.

In Formula XI, a drug can be a therapeutically active agent linkeddirectly or through a linker.

Formula XII is a cationic guggul derivative, wherein R₆, R₇, and R_(s)are same or different and are hydrogen, methyl, alkyl, substitutedalkyl, alkyoxy, substituted alkyloxy groups, optionally hydroxylated,aminated, or polyaminated having overall positive charge.

Guggul derivatives (III, XII), guggulphospholipids (III-X) andguggul-drug conjugates (XI) in the present invention can be in any formof geometric isomers. For example Formulas can be in the form ofE-isomer or Z-isomer or mixture of E and Z isomers. Formulas III-XII inpresent invention can also be optically pure or mixture of opticalisomers.

One embodiment of the present invention is set forth in FIG. 1, whichshows novel approaches to the synthesis of guggul derivative (III, XII),guggulphospholipids IV-X, and guggul-drug conjugate (XI) starting fromguggulsterol (IIA). Guggul derivative, guggulphospholipids andguggul-drug conjugate of Formulas III-XII can be prepared by any desiredmethod, and the invention provides methods of preparing guggulderivatives, guggulphospholipids and analogues thereof. The method ofpreparing compounds from guggulsterol IIA in the present invention canbe done in single step or a sequence of many steps.

In accordance with the inventive method, guggul derivatives of FormulaIII in the present invention can be prepared by the reaction ofguggulsterol IIA with acyl chlorides in the presence of base in asuitable solvent. Specific examples of bases include but not limited topyridine, triethylamine, diisopropylethylamine, diisopropylamine,butylamine and the like. Examples of suitable solvents include but notlimited to, pyridine, triethylamine, dichloromethane, chloroform,tetrahydrofuran (THF), diethyl ether, t-butyl methyl ether, hexane, 1,dichloroethane, benzene, toluene, N,N-dimethylformamide (DMF) and thelike.

Another method of the present invention involves methods of preparingguggul derivatives of formula III by the reaction of guggulsterol IIAwith fatty acids and N,N′-dicyclohexyl carbodimide (DCC) or1-ethyl-3-(3-dimethylaminopropyl)-carbodimide (EDC) in the presence ofN,N′-dimethylaminopyridine (DHAP) in a suitable solvent. Examples ofsuitable solvents include but not limited to, dichloromethane,chloroform, tetrahydrofuran (THF). 1,2-dichloroethane, benzene, toluene,pyridine, N, N-dimethylformamide (DMF) and the like

Another embodiment of the present invention involves methods ofpreparing Guggul phospholipids of Formula IV by reacting gugguisterolIIA with phosphoramidite reagent in an inert solvent (for exampledichloromethane and the like) in presence of base (for exampleN,N-diisopropylethylamine or the like), then with alcohol (ROH) inpresence of an activator such as 1H-tetrazole or the like followed byoxidation with tart-butylhydroperoxide or the like. The deprotection canbe achieved by any suitable method, selected according to the protectinggroup present on the phosphate group. For example a methyl group can beremoved with sodium iodide or trimethylamine, a benzyl group can beremoved with sodium iodide or catalytic hydrogenolysis, cyanoethyl andfluroenylmethyl groups by the treatment with a tertiary base such astriethylamine, a silyl group can be deprotected with fluoride ion oracidic medium.

Examples of suitable phosphoramidite reagents in the present inventioninclude but are not limited to N,N-diisopropylmethylphosphoramidicchloride, (Bruzik et al, Tet. Lett. 1995, 36, 2415-2418;,(benzyloxy)(N,N-diisopropylamino)chlorophosphine (see, e.g., Prestwichet al, J. Am. Chem. Soc. 1991, 113, 1822-1825), benzyloxybis(diisopropylamino) phosphine (see, e.g., Dreef et al. Tetrahedron Lett.1988, 29, 6513-6516), 2-cyanoethyl-N,N-tetraisopropylphosphoramidite(see, e.g., Browne et al. J Chem. Soc. Perkin Trans. 1.

Examples of other suitable phosphorylating reagents in the presentinvention include but are not limited to 2-bromoethyldichlorophosphate(Wissner, A. et al, J. Med. Chem. 1986. 29, 1315-1319),trimethylsilylethyl dicholorphosphate (Martin, S. F. et al, J Org. Chem.1994, 59, 4805-4820), methyl dichlorophosphate (Andresen, T. L. et al,J. Med. Chem. 2005, 48, 7305-7314),2-chloro-2-oxo-1,3,2-dioxaphospholane (Marx, M. H. et al, J. Med. Chem.1988, 31, 858-863), 2-chloropheny dichlorophosphate phosphorusoxychloride (Andresen, T. L. et al, J. Med. Chem. 2004, 47, 1694-1703).

Examples of suitable alcohols include but are not limited to alcohols ofthe formula ROH wherein R is a saturated or unsaturated acyl or alkylgroup having between 1 and 34 carbon atoms. The terms “alkyl”encompasses saturated or unsaturated straight chain and branched-chainhydrocarbon moieties. The term “substituted alkyl” comprises alkylgroups further bearing one or more substituents selected from hydroxyl,alkoxy (of a lower alkyl group), mercapto (of a lower alkyl group),cycloalkyl, substituted cycloalkyl, halogen, cyano, nitro, amino, amino,imino, thio, —C(O)H, acyl, oxyacyl (of a lower acyl) carboxyl and thelike.

Another embodiment of the present invention involves a method ofpreparation of guggulphospholipids of Formula V by reacting guggulsterolIIA with a. phosphoramidite reagent in an inert solvent (for example,dichloromethane and the like) in presence of base (for example,N,N-diisopropylethylamine or the like), then with 1,2-disubstitutedglycerol in presence of an activator such as, e.g., 1H-tetrazole or thelike, followed by oxidation with tert-butylhydroperoxide or the like.The deprotection can be achieved by any suitable method, depending onthe particular protecting group present on the phosphate group. Methodsfor deprotection are well known to those skilled in the art. Forexample, a methyl group can be removed with sodium iodide ortrimethylamine, a benzyl group can be removed with sodium iodide orcatalytic hydrogenolysis, cyanoethyl and fluroenylmethyl groups by thetreatment with a tertiary base such as triethylamine, and a silyl groupcan be deprotected with fluoride ion or acidic medium. Alternatively,1,2-disubstituted glycerol can be first reacted with the phosphoramiditereagent and subsequently with guggulsterol IIA.

Another method in the present invention for the preparation of FormulasIV and V involves reaction of alcohol (ROH) or 1,2-disubstitutedglycerol, respectively, with o-chlorophenyl dichlorophosphate (CPDCP)and, subsequently, with guggulsterol IIA in an inert solvent (forexample, dichloromethane and the like) in the presence of base (forexample, pyridine and the like) to provide the respective intermediates.The removal of o-chlorophenyl can be accomplished by reaction ofrespective intermediates with 2-pyridinealdoxime (PAO) and1,1,3,3-tetramethylguanidine (TMG) to give IV and V respectively.Besides 2-pyridinealdoxime (PAO), other reagents such as2-nitrobenzaldoxime in the presence of TMG can be used for the removalof o-chlorophenyl group.

Another method o the present invention for the preparation of FormulasIV and V involves reaction of alkyl phosphatidic acid or 1,2-disubstituted-sn-glycero-3-phosphatidic acid, respectively, with 1,2-dicyclohexylcarbodimide

(DCC) and, subsequently, with guggulsterol IIA in an inert solvent (forexample, dichloromethane and the like) in the presence of base (forexample dimethylaminopyridine, DMAP) to provide IV and V respectively.Different salts of IV and V can be obtained by treating with appropriatebases. For example, treatment with dilute sodium hydroxide will providesodium salt of IV and V, dilute potassium hydroxide will providepotassium salt of Formula IV and V, and dilute ammonium hydroxide willprovide ammonium salt of Formula IV and V.

Yet another method of preparing guggulphosphate of Formula VI can be byreacting guggulsterol IIA with phosphorus oxychloride in presence ofbase (for example, pyridine and the like) in a suitable solvent (such asdichloromethane and the like) and later treating with water. Differentsalts of VI can be obtained by treating with appropriate bases. Forexample, treatment with dilute sodium hydroxide will provide sodium saltof VI, dilute potassium hydroxide will provide potassium salt of FormulaVI, and dilute ammonium hydroxide will provide ammonium salt of FormulaVI.

In another embodiment of the present invention, guggulsulfate of FormulaVII is synthesized by reacting guggulsterol IIA with chlorosulfonic acidin presence of base (for example, pyridine and the like) in a suitablesolvent (such as dichloromethane and the like). Different salts of VIIcan be obtained by treating with appropriate bases. For example,treatment with dilute sodium hydroxide will provide sodium salt of VII,dilute potassium hydroxide will provide potassium salt of Formula VII,and dilute solution of ammonium hydroxide will provide ammonium salt offormula VII.

In another embodiment of the present invention guggulphospholipid ofFormula VIII (R₃, R₄, R₅=H) is prepared by the reaction of gugguisterolIIA with trimethylsilylethyl dicholorphosphate in presence of base (forexample N,N-diisopropylethylamine or the like) in an inert solvent (forexample tetrahydrofuran or the like) then with N-(tert-butoxycarbonyl)ethanolamine followed by oxidation with tert-butylhydroperoxide or thelike. The deprotection of Cert-butoxycarbonyl (Hoc) group andtrimethylsilylethyl group can be done under acidic condition (forexample with trifluoroacetic acid and the like.

Yet another method for preparing Formula VIII (R₃, R₄, R₅=H) in thepresent invention involves reacting guggulsterol IIA with methyldichlorophosphate in presence of base (for example triethylamine) in aninert solvent followed by reaction with N-(cert-butoxycarbonyl)ethanolamine. Deprotection of phosphate methyl group can be achievedwith sodium iodide or trimethylamine.

Another embodiment of the present invention involves method ofpreparation of Formula VIII (R₃, R₄, R₅=CH₃) in the present inventioncan be synthesized by the reaction of guggulsterol IIA with2-bromoethylchloro phosphate in presence of base (for example,triethylamine or pyridine and the like) followed by treatment withtrimethylamine in suitable solvent such as chloroform and the like.

Another method of the preparation of Formula VIII (R₃, R₄, R₅=CH₃)involves reaction of guggulsterol IIA with2-chloro-2-oxo-1,3,2-dioxaphospholane and subsequently reacting withtrimethylamine in an inert solvent (for example, acetonitrile and thelike).

Yet another method of preparing VIII (R₃, R₄, R₅=CH₃) involves reactionof guggulsterol IIA with phosphoramidite reagent in an inert solvent(for example dichloromethane and the like) in presence of base (forexample N,N-diisopropylethylamine or the like), then with cholinechloride in presence of an activator such as 1H-tetrazole or the likefollowed by oxidation with tert-butylhydroperoxide or the like. Thedeprotection can be achieved by any suitable method depending on theprotecting group present on the phosphate group. For example a methylgroup can be removed with sodium iodide or trimethylamine, a benzylgroup can be removed with sodium iodide or catalytic hydrogenolysis,cyanoethyl and fluroenylmethyl groups by the treatment with a tertiarybase such as triethylamine; a silyl group can be deprotected withfluoride ion or acidic medium.

Formula VIII in the present invention can be obtained by another methodof preparation in which guggulsterol IIA is reacted with phosphorusoxychloride in presence of base (for example, triethylamine and thelike) and later with choline tosylate in suitable solvent such aspyridine and the like.

A method of preparing guggulphospholipids of Formula IX in the presentinvention involves reaction of guggulsterol with phosphoramidite reagentin the presence of base (for example, N,N-diisopropylethylamine and thelike) in an inert solvent (for example, dichloromethane and the like)followed by reaction with 1,2-isopropylidene glycerol. Theisopropylidene group can later be removed under acidic condition (suchas HCl and the like) while the protecting groups on the phosphate can beachieved by any suitable method depending on the protecting grouppresent on the phosphate group. For example a methyl group can beremoved with sodium iodide or trimethylamine, a benzyl group can beremoved with sodium iodide or catalytic hydrogenolysis, cyanoethyl andfluroenylmethyl groups by the treatment with a tertiary base such astriethylamine; a silyl group can be deprotected with fluoride ion oracidic medium.

Formula X in the present invention is prepared by reacting guggulsterolIIA with methyl chlorophosphate in presence of base (for exampletriethylamine) in an inert solvent followed by reaction withN-(tert-butoxycarbonyl) ethanolamine to provide an intermediate whichwas subsequently reacted with mPEG-succinimidyl carbonate (SC-PEG).Deprotection of phosphate methyl group can be achieved with sodiumiodide or trimethylamine.

Another embodiment of the present invention is to prepare guggul-drugconjugate having Formula XI as a prodrug by conjugating a drug withguggulsterol directly or through a linker.

Yet another embodiment of the present invention is to prepare cationicguggul derivatives (positively charged guggul derivatives) havingformulas VIII (X=methyl group) and XII from guggulsterol IIA orguggulsterone I.

Guggulsterol in the present invention can be obtained commercially orsynthesized as per the literature procedure. Guggulsterol in the presentinvention can be pure isomer or mixture of isomers. For example,guggulsterol can be Z-guggulsterol or E-guggulsterol or mixture ofE-guggulsterol and Z-guggulsterol. Guggulsterols can be optically pureor mixture of optical isomers.

Another embodiment of the present invention, is represented in FIG. 2,in which guggulsterol is synthesized starting from guggulsterone I.Guggulsterone I on reduction with 9-borabicyclo [3.3.1] nonane (9-BBN)in suitable solvent (for example, methylene chloride, tetrahydrofuran orthe like) will provide of IIA, IIB, or a mixture IIA and IIB. Duringreduction, it is conceivable that some quantities of Formulas IIC, IID,IIE and IIF will formed as minor products in addition to IIA and IIBduring the reduction. Compound IIA can be isolated by columnchromatography. Compound IIE on further oxidation with suitableoxidizing agent, for example, manganese dioxide (MnO2) in suitablesolvent such as dichloromethane will provide compound IIA, IIB ormixture of IIA and IIB.

Another embodiment of the present invention, is represented in FIG. 2,in which guggulsterol is synthesized starting from guggulsterone I.Guggulsterone I on reduction with lithium aluminum hydride or sodiumborohydride in suitable solvent (for example, methylene chloride,tetrahydrofuran or the like) provide of IIA, IIB, or a mixture IIA andIIB. During reduction, it is conceivable that sonic quantities ofFormulas IIC, IID, IIE and IIF will formed as minor product in additionto IIA and IIB during the reduction. Compound IIA can be isolated bycolumn chromatography. Compound IIE on further oxidation with suitableoxidizing agent, for example, manganese dioxide (MnO2) in suitablesolvent such as dichloromethane will provide compound IIA, IIB ormixture of IIA and IIB.

The term “prodrug” is defined as a pharmacologically inactive compoundthat is converted into an active agent by metabolic transformation. Theobjective is to chemically modify the drug (into its prodrug) in orderto render it temporarily inactive. In vivo, via the action of enzyme(s),this prodrug then decomposes thereby liberating the active principle. Inmost cases, a judiciously selected chemical group is bound covalently tothe active principle. This group will often govern the solubility of theprodrug, its stability, and the rate at which it liberates the activeprinciple and the particular enzyme required for its transformation(Malet-Martino et al. Curr. Med. Chem.-Anti-Cancer Agents, 2002, 2,267-310)

The term “linker” is defined herein as a group or chain containing oneor more functional group for covalent binding with the lipid carrier andbiologically active nucleoside. Preferred embodiments comprise a linkerhaving at least two functional groups, wherein the linker has a firstend and a second end and wherein the lipid is attached to the first endof the linker through a first linker functional group and the nucleosideis attached the second end of the linker through a second linkerfunctional group. These groups can be designated either as weak orstrong, based on the stability of the covalent bond which the linkerfunctional group will form between the linker and either the lipidcarrier or the biologically-active nucleoside. The weak functionalitiesinclude, but are not limited to, phosphoramidite, phosphoesters (such asphosphodiester, phosphotri ester and phosphonate), carbonate, amide,carboxyl-phosphorl anhydride, ester and thioester. The strongfunctionalities include, but are not limited to, ether, thioether,amine, amide, and ester. The use of a strong linker functional groupbetween the linker and the nucleoside will tend to decrease the rate atwhich the compound will be released in vivo, whereas the use of a weaklinker functional group between the linker and the nucleoside may act tofacilitate in vivo release of the compound. In preferred embodiments,each of the first and second functional linker groups is a hydroxylgroup, a primary or secondary amino group, phosphate group orsubstituted derivative thereof, a carboxylic acid, carbonate, carbamateor carbonyl group. The “linker” herein also comprises, in addition tothe functional groups at either end, (CH₂), groups (where n=0-20) in thecenter optionally substituted with functional groups, such as alkyl,alkoxy, hydroxyl, carbonyl, carboxyl, carbamate, aldehyde, amino, halo,polyalkoxy, PEG groups, phosphate, phosphonate and pyrophosphate groups.

The inventive method can be used to prepare pharmaceutically activeguggulphospholipids, their cationic and anionic analogues, their fattyacid analogues and variants comprising fatty acid/alkyl chain of varyinglength and saturation/unsaturation. The general structure ofphospholipids comprises a fatty acid hydrocarbon chain and a carboxylicacid group. In general, the length of fatty acid hydrocarbon chainranges from

about 2 to about 34 carbons and can be saturated or unsaturated.However, the carbon chain is more typically between about 4 carbons andabout 24 carbon atoms. In some embodiments, it is desirable for thehydrocarbon chain to comprise, for example, at least 6 carbon atoms orat least about 12 carbon atoms or at least about 14 carbon atoms.Typically, the length of fatly acid hydrocarbon is less than about 24carbon atoms, or even less than about 20 carbon atoms. Indeed, ananalogue of the present invention containing fatty acids/alkyl chains ofintermediate length can also be prepared by the inventive method.

The term ‘phosphate protecting group’ used herein the invention refersto the commonly used protecting groups described by T. W. Greene and P.G. Wuts, Protective Groups in Organic Synthesis, 3 rd edition, JohnWiley &. Sons, New York (1999). Such protecting groups include alkylphosphates including methyl, ethyl, cyclohexyl, t-butyl; 2-substitutedethyl phosphates including 2-cyanoethyl, 4-cyano-2-butenyl,2-(methyldiphenylsilypethyl, 2-(trimethylsilypethyl,2-(triphenylsilypethyl; haloethyl phosphates including2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,2,2-trifluoroethyl; benzylphosphates including 4-chlorobenzyl, fluorenyl-9-methyl, diphenylmethyland amidates.

Phospholipids fatty acid typically are classified by the number ofdouble or triple bonds in the hydrocarbon chain (unsaturation).Asaturated fatty acid does not contain any double or triple bonds, andeach carbon in the chain is bond to the maximum number of hydrogenatoms. The degree of unsaturation of fatty acid depends on the number ofdouble or triple bonds in the hydrocarbon chain. In this respect, amonounsaturated fatty acid contains one double bond, whereas apolyunsaturated fatty acid contains two or more double bonds [e.g.,Oxford Dictionary of Biochemistry and Molecular Biology, rev .ed., A. D.Smith (ed.), Oxford University Press (2000), and Molecular Biology Ofthe Cell, 3rd ed,, B. A. Alberts(ed), Garland Publishing, New York(1994)].Fatty acids of varying chain length and unsaturation can be usedto prepare compounds of present invention. Preferred fatty acids rangefrom carbon chain length of about 2 to 34, preferably between about 4and about 24 carbons or even less than about 20 carbon atoms, andinclude tetranoic acid(C4:0),pentanoic acid(C5:0),hexanoicacid(C6:0),heptanoic acid(C7:0),octanoic acid(C8:0),nonanoicacid(C9:0),decanoic acid(C10:0),undecanoic acid(C11:0),dodecanoicacid(C12:0),tridecanoic acid(C13:0),tetradecanoic(myristic)acid(C14:0),pentadecanoic acid(C15:0),hexadecanoic (palmatic) acid(C16:0),heptadecanoic acid(C17:0),octadecanoic(stearic)acid(C18:0),nonadecanoicacid(C19:0),eicosanoic(arachidic)acid(C20:0),heneicosanoicacid(C21:0),docosanoic (behinic)acid(C22:0),tricosanoicacid(C23:0),tetracosanoic acid(C24:0),10-undecanoicacid(C11:1),11-dodecanoic acid(C12:1),12-tridecanoicacid(C13:1),myristoleic acid(C14:1),10-pentadecaenoicacid(C15:1),palmitoleic acid(C16:1),oleic acid(C18:1),linoleic acid(C18:2),linolenic acid(C18:3),eicosenoic acid (C21:1),eicosadienoic acid(C20:2), eicosatrienoic acid (C20:3), arachidonic acid(cis-5,8,11,14-eicostetraenoic acid), and cis-5, 8, 11, 14,17-eicospentaenoic acid. Other named fatty acids can also be used.Examples of such include saturated fatty acids such as ethanoic(oracetic)acid, propanoic(or propionic)acid,butanoic(orbutyric)acid,hexacosanoic(or cerotic)acid, octacosanoic(ormontanic)acid,tricontanoic(or melissic)acid, dotricontanoic(orlacceroic) acid, tetratricontanoic(or gheddic)acid,pentatricontanoic(orceroplastic)acid, and the like; mono-unsaturated fatty acids such astrans-2-butenoic(or crotonic)acid,cis-2-butenoic(or isocrotonoic)acid,2-hexenoic(or isohydrosorbic)acid,4-decanoicobtusilic)acid,9-decanoic(or caproleic)acid,4-dodecenoic(orlinderic)acid,5-dodecenoic (or denticetic)acid,9-dodecenoic(lauroleic)acid,4-tetradecenoic(ortsuzuic)acid,5-tetradecenoic(or physeteric)acid,6-octadecenoic orpetroselenic)acid,trans-9-octadecenoic(or elaidic)acid,trans-11-octadecenoic(or vaccinic)acid,9-eicosenoic or gadoleicacid,11-docosenoic(or cetoleic)acid,13-decosenoic(orerucic)acid,15-tetracosenoic(or nervonic)acid,17-hexacosenoic(orximenic)acid,21-triacontenoic(or luniequeic)acid, and the like;dienoicunsaturated fatty acids such as 2,4-pentadienoic(orβ-vinylacrylic)acid,2,4-hexadienoic(or sorbic)acid,2,4-decadieno c(orstillingic)acid; 2,4-dodecadienoic acid; 9,12-hexadecadienoic acid;cis-9-cis-octadecadienoic(or α-linoleic)acid;trans-10-trans-12-octadecadienoic(or linoleaidic)acid;trans-11-trans-12-octadecadienoic acid; 11,14-eicosadienoicacid;13,16-docosadienoic acid;17,20-Hexacosadienoic acid and the like;trienoic unsaturated fatty acids such as 6,10,14-hexadecatrienoic (orhiragonic) acid; 7,10,13-hexadecatrienoic acid;cis-6-cis-9-cis-12-Octadecatrienoic (or γ-linoleic) acid;trans8-trans-10-trans-12-octadecatrienoic (or β-calendic) acid;cis-8-trans-10-trans-12-octadecatrienoic (or β-calendic) acid;cis-8-trans-10-cis-12-octadecatrienoicacid;cis-9-cis-12-cis-15-octadecatrienoic(or α-linolenic)acid;trans-9-trans-12-trans-15-octadecatrienoic(orα-linolenelaidic)acid;cis-9-trans-11-trans-13-octadecatrienoic(orα-eleostearic)acid;trans-9-trans-11-trans-13-octadecatrienoic(orβ-eleostearic)acid;cis-9-trans-11-cis-13-octadecatrienoic(or punicic)acid;5,8,11-eicosatrienoic acid;8,11,14-eicosatrienoic acid and thelike; tetraenoic unsaturated fatty acids such as4,8,11,14-hexadecatetraenoic acid;6,9,12,15-hexadecatetraenoicoctadecatetraenoic(or oroctic)acid;6,9,12,15-octadecatetraenoicacid;9,11,13,15-octadecatetraenoic(or α- or β-parinaric)acid;9,12,15,18-octadecatetraenoic acid;4,8,12,16-eicosatetraenoicacid;6,10,14,18-eicosatetraenoic acid;4,7,10,13-docastetraenoicacid;7,10,13,16-docosatetraenoic acid;8,12,16,19-docosatetraenoic acidand the like; penta-and hexaenoic unsaturated fatty acids such as4,8,12,15,18-eicospentaenoic(ortimnodonic)acid;4,7,10,13,16-docospentaenoicacid;4,8,12,15,19-docospentaenoic(or clupanodonic)acid;7,10,13,16,19-docosapentaenoic acid;4,7,10,13,16,19-docosahexaenoicacid;4,8,12,15,18,21-tetracosahexaenoic(or nisinic)acid and the like;branched chain fatty acids such as 3-methylbutanoic(orisovaleric)acid;8-methyldodecanoic acid;10-methylundecanoic(orisolauric)acid;11-methyldodecanoic(orisoundecylic)acid;12-methyltridecanoic (orisomyristic)acid;13-methyltetradecanoic(or isopentadecylic)acid;14- methylpenta-decanoic(or isopalmitic)acid;15-methylhexadecanoic acid;10-methylheptadecanoicacid;16-methylheptadecanoic(or isostearic)acid;18-methylnonadecanoic(orisoarachidic)acid;20-methylheneeicosanoic(orisobehenic)acid;22-methvltricosanoic(orisolignoceric)acid;24-methylpentacosanoicisocerotic)acid;26-methylheptacosanoic(orisomonatonic)acid;2,4,6-trimethyloctacosanoic(or mycoceranic ormycoserosic)acid;2-methyl-cis-2-butenoic9angelic)acids;2-methyl-trans-2-butenoictigilic)acid;4-methyl-3-pentenoic(or pyroterebic)acid, and the like.

Guggulphospholipids of the present invention can conveniently beincorporated into liposomes, emulsion, micelles, vesicles or complexes.Such liposome can also include one or more therapeutic agent. Suchformulations according to the present invention can be prepared by anysuitable technique. The invention provides a method for preparing aliposome, emulsion, micelles, vesicles, complexes or other lipidformulation comprising a novel guggulphospholipid or other bioactivemolecules of the present invention.

The liposome composition or complexes including micelles, vesicles, andemulsions can also include other lipids, for example, the compositioncan include one or more natural or synthetic phospholipid. Thephospholipids will be selected from the group consisting ofphosphatidylcholine, soy phosphatidylcholine, hydrogenated soyphosphatidylcholine and mixture thereof, egg phosphatidylcholine,dilauryloylphosphatidylcholine, dimyristoylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,dioleylphosphatidylcholine, dielaidoylphosphatidylcholine,diarachidonoylphosphatidylcholine and mixture thereof in different molarratio, phosphatidylethanolamine, sphingomyelin, dimyristoylphosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine,brainsphingomyelin, dipalmitoyl sphingomyelin, disteroyl sphingomyelinand mixtures thereof. The novel guggulphospholipid of the presentinvention comprises guggulsterones or guggulsterol, which can serve assubstitute of cholesterol or derivatives of cholesterol. Alternativelyor additionally, the composition can include one or more sterols ifnecessary such as, cholesterol, derivatives of cholesterol, cholesterylesters, coprostanol, cholestanol, cholestane, cholesterol hemisuccinate,cholesterol sulfate, guggulsterols, and derivatives of guggulsterols,guggulsulfate, guggulsteryl esters, and mixtures thereof.

The liposome composition or complexes including micelles, vesicles andemulsions can also include one or more negatively charged phospholipids.The negatively charged phospholipids will be selected from the groupconsisting of phosphatidylglycerol, phosphatidylserine, phosphatidicacid, poly(ethylene glycol)-phosphatidylethanolamine,dilauroylphosphatidyl glycerol, dimyristoylphosphatidyl glycerol,dipalmitoylphosphatidyl glycerol, distearoylphosphatidyl glycerol,dioleoylphosphatidyl glycerol, dimyristoyl phosphatidic acid,dipalmitoyl phosphatidic acid, dimyristoyl phosphatidyl serine,dipalmitoyl phosphatidyl serine, brain phosphatidyl serine, and mixturesthereof.

The liposome composition or complexes including micelles, vesicles andemulsions can also include one or more positive charge lipids. Examplesof positive charge lipids in the present invention include compounds offormula XII and VIII (where X is a methyl group). The liposome orcomplexes may further contain phospholipids such as phosphatidylcholine,phosphatidylglycerol and cholesterol or cholesterol derivative incertain ratio such that the said liposome or complexes have a. netoverall positive charge.

The liposome composition or complexes including micelles, vesicles andemulsions can also include stabilizers, absorption enhancers,antioxidants, biodegradable polymers and medicinally active agents amongother ingredients. In some embodiments, it is preferable for theinventive composition, especially liposome composition, to include oneor more targeting agents, such as carbohydrate or protein or otherligand that binds to a specific substrate, for example, that recognizecellular receptors. The inclusion of such agents, such as carbohydrateor one or more proteins selected from groups of proteins consisting ofantibodies, antibody fragments, peptides, peptide hormones, receptorligands such as an antibody to a cellular receptor and mixtures thereof,can facilitate targeting a liposome to a predetermined tissue or celltype.

The liposome composition or complexes including micelles, vesicles andemulsions can also include one or more active agents for the medicinaluse. A single active agent can be included, or a mixture of activeagents (e.g., two or more active agents) can be included within thecomposition. Active agents (or “drugs”) can be present in any suitablemanner in the composition. For example, they can be complexes with thepharmaceutically active guggulphospholipids of the present invention andtheir analogues in the composition. Additionally, or alternatively, oneor more active agents can be entrapped within liposome, when thecomposition is a liposome composition,

Drugs, active agents or therapeutic agents that are compatible with thepresent invention include, for example, agents which act on theperipheral nerves, adrenergic receptors, cholinergic receptors, theskeletal muscles, the cardiovascular system, smooth muscles, the bloodcirculatory system, synaptic sites, neuroeffector functional sites,endocrine and hormone systems, the immunological system, thereproductive system, the skeletal system, the alimentary and excretorysystems, the histamine system and the central nervous system. Suitableagents may be selected from, for example, proteins, enzymes, andhormones, nucleotides (including sense and antisense oligonucleotides)(e.g., U.S. Pat. No. 6,126,965, 2000), polynucleotide, nucleoproteins,polysaccharides, glycoproteins, lipoproteins, polypeptides, steroids.Active agents can be analgesics, anesthetics, anti-arrhythmic agents,antibiotics, antiallergic agents, antifungal agents, anticancer agents,anticoagulants, antibodies, cytoxines, doxorubicin, etopside,derivatives of the foregoing. Additional examples of drugs which may bedelivered according to the method include, prochlorperzine edisylate,ferrous sulfate, aminocaproic acid, mecamylamine hydrochloride,procainamide hydrochloride, amphetamine sulfate, methamphetaminehydrochloride, benzatnphetaniine hydrochloride, isoproterenol sulfate,phenmetrazine hydrochloride, bethanechol chloride, methacholinechloride, pilocarpine hydrochloride, atropine sulfate, scopolaminebromide, isopropamide iodide, tridihexethyl chloride, phenforminhydrochloride, methylphenidate hydrochloride, theophylline cholinate,cephalexin hydrochloride, diphenidol, meclizine hydrochloride,prochlorperazine maleate, phenoxybenzamine, thiethylperzine maleate,anisindone, diphenadione erythrityl tetra nitrate, digoxin,isoflurophate, acetazolamide, methazolamide, bendroflumethiazide,chloropromaide, tolazamide, chlormadinone acetate, phenaglycodol,allopurinol, aluminum aspirin, methotrexate, acetyl sulfisoxazole,erythromycin, hydrocortisone, hydrocorticosterone acetate, cortisoneacetate, dexamethasone and its derivatives such as betamethasone,triamcinolone, methyl testosterone, 17-β-estradiol, ethinyl estradiol,ethinyl estradiol 3-methyl ether, prednisolone, 17-α-hydroxyprogesteroneacetate, 19-norprogesterone, norgestrel, norethindrone, norethisterone,norethiederone, progesterone, norgesterone, norethynodrel, aspirin,indomethacin, naproxen, fenoprofen, sulindac, indoprofen, nitroglycerin,isosorbide dinitrate, propranolol, timolol, atenolol, alprenolol,cimetidine, clonidine, imipramine, levodopa, chlorpromazine, methyldopa,dihydroxyphenylalanine, theophylline, calcium gluconate, ketoprofen,ibuprofen, cephalexin, erythromycin, haloperidol, zomepirac, ferrouslactate, vincamine, diazepam, phenoxyhenzamine, diltiazem, milrinone,mandol, quanbenz, hydrochlorothiazide, ranitidine, flurbiprofen,fenufen, fluprofen, tolmetin, alclofenac, mefenamic, flufenamic,difuinal, nimodipine, nitrendipine, nisoldipine, nicardipine,felodipine, lidoflazine, tiapamil, gallopamil, amlodipine, mioflazine,lisinolpril, enalapril, enalaprilat captopril, ramipril, famotidine,nizatidine, sucralfate, etintidine, tetratolol, minoxidil,chlordiazepoxide, diazepam, amitriptyline, and imipramine. Furtherexamples are proteins and peptides which include, but are not limitedto, bone morphogenic proteins, insulin, colchicine, glucagon, thyroidstimulating hormone, parathyroid and pituitary hormones, digestivehormones, calcitonin, rennin, prolactin, corticotrophin, thyrotropichormone, follicle stimulating hormone, chorionic gonadotropin,gonadotropin releasing hormone, bovine somatotropin, porcine

Generally, liposome or complexes including micelles, vesicles andemulsions can have a net neutral, negative or positive charge. Forexample, positive liposome can be formed from a solution containingnatural or synthetic phosphatidylcholine, cationic phosphatidylcholineof the present invention with or without cholesterol and enoughstearylamine to overcome the negative charge if any. Negative liposomecan be formed from solutions containing natural or syntheticphosphatidylcholine, cholesterol, and phosphatidylglycerol or negativelycharged phosphatidylcholine variants prepared by the methods describedherein.

The liposome or complexes including micelles, vesicles and emulsions ofthe present invention can be multi or unilamellar vesicles depending onthe particular composition and procedure to make them. Liposome orcomplexes including micelles, vesicles and emulsions can be prepared tohave substantially homogeneous sizes in a selected size range, such asabout 20 micron or less, or about 10 micron or less, or about 5 micronor less, or aboutl micron or less, or about 500 nm or less, about 200nmor less, or about 100nm or less. One effective sizing method involvesextruding an aqueous suspension of the liposome through a series ofpolycarbonate membranes having a selected uniform pore size; the poresize of the membrane will correspond roughly with the largest sizes ofliposome produced by extrusion through that membrane.

The liposome composition or complexes including micelles, vesicles andemulsions can be in any desired form. For example, for pharmaceuticaluse, the composition can be ready for administration to a patient.Alternatively, the composition can be in dried or lyophilized form.Where the composition is dried or lyophilized, preferably thecomposition includes a cryoprotectant as well. Suitable cryoprotectantsinclude, for example, sugars such as trehalose, maltose, lactose,sucrose, glucose, and dextran, with the most preferred sugars from aperformance point of view being trehalose and sucrose. Other morecomplicated sugars can also be used, such as, for example, aminoglycosides, including streptomycin and dihydrostreptomycin.

Any suitable method can be employed to form the liposome or complexesincluding micelles, vesicles and emulsions. For example, lipophilicliposome-forming or lipophilic complex-forming ingredients, such asnatural or synthetic phosphatidylcholine, pharmaceutically activeguggulphospholipids of the present invention, with or withoutcholesterol or cholesterol derivatives, and α-tocopherol can bedissolved or dispersed in a suitable solvent or combination of solventsand dried. Suitable solvents include any ionic solvent or any non-polaror slightly polar solvent, such as t-butanol, ethanol, methanol,chloroform, or acetone that can be evaporated without leaving apharmaceutically unacceptable residue. Drying can be by any suitablemeans such as by lyophilization. The dehydration is typically achievedunder vacuum and can take place either with or without prior freezing ofthe liposome preparation. Hydrophilic ingredients can be dissolved inpolar solvents, including water.

Mixing the dried lipophilic ingredients with the hydrophilic mixture canform liposome or complexes. Mixing the polar solution with the dry lipidfilm can be by any means that strongly homogenizes the mixture.Vortexing, magnetic stirring and/or sonicating can effect thehomogenization.

Where active agents (or a mixture of active agents) are included in theliposome or complexes, the invention provides a method for retaining adrug in a liposome or complexes. The pharmaceutically activeguggulphospholipids of the present invention, their analogues or otherbioactive molecules as described herein and a drug or drugs (e.g., anactive agent or a mixture of active agents) is included within aliposome or a complex. For example, active agent(s) can be dissolved ordispersed in a suitable solvent and added to the liposome mixture priorto mixing. Typically hydrophilic active agents will be added directly tothe polar solvent and hydrophobic active agents will be added to the nonpolar solvent used to dissolve the other ingredients but this is notrequired. The active agent could be dissolved in a third solvent (e.g.,ionic solvent or solvent mix and added to the mixture of polar solventwith the lipid film prior to homogenizing the mixture.

Liposome or complexes including micelles, vesicles and emulsions can becoated with biodegradable polymers such as sucrose, epichlorohydrin,branched hydrophilic polymers of sucrose, polyethylene glycols,polyvinyl alcohols, methoxypolyethylene glycol, ethoxypolyethyleneglycol, polyethylene oxide, polyoxyethylene, polyoxypropylene, celluloseacetate, sodium alginate, N,N-diethylaminoacetate, block copolymers ofpolyoxyethylene and polyoxypropylene, polyvinyl pyrrolidone,polyoxyethylene X-lauryl ether wherein X is from 9 to 20, andpolyoxyethylene sorbitan esters.

Antioxidants can be included in the liposome composition, complexes orother lipid composition. Suitable antioxidants include compounds such asascorbic acid, tocopherol, and deteroxime mesylate.

Absorption enhancers can be included in the liposome composition,complexes or other lipid composition. Suitable absorption enhancersinclude sodium salicylate-chenodeoxy cholate, sodium deoxycholate,polyoxyethylene 9-lauryl ether, chenodeoxy cholate-deoxy cholate andpolyoxyethylene 9-lauryl ether, monoolein, sodiumtauro-24,25-dihydrofusidate, sodium taurodeoxycholate, sodiumglycochenodeoxycholate, oleic acid, linoleic acid, linolenic acid.Polymeric absorption enhancers can also be included such aspolyoxyethylene ethers, polyoxyethylene Sorbian esters, polyoxyethylene10-lauryl ether, polyoxyethylene 16-lauryl ether, atone(1-dodcylazacycloheptane-2-one).

The inventive lipid (and their liposome or complexes) composition alsocan include one or more pharmaceutically acceptable excipients. Forexample, pharmaceutically suitable excipients include solid, semi-solidor liquid diluents, fillers and formulation auxiliaries of all kinds.The invention also includes pharmaceutical preparations in dosage units.This means that the preparations are in the form of individual parts,for example vials, syringes, capsules, pills, suppositories, orampoules, of which the content of the liposome or complex formulation ofactive agent corresponds to a fraction or a multiple of an individualdose. The dosage units can contain, for example, 1, 2, 3, or 4individual doses, or ½, ⅓, or ¼ of an individual dose. An individualdose preferably contains the amount of active agent which is given inone administration and which usually corresponds to a whole, a half, athird, or a quarter of a daily dose.

Tablets, dragees, capsules, preferably enteric coated capsules ortablets, pills, granules, suppositories, solutions, suspensions,emulsions, pastes, ointments, gels, creams, lotions, powders and sprayscan be suitable pharmaceutical preparations. Suppositories can contain,in addition to the liposome active agent, suitable water-soluble orwater-insoluble excipients. Suitable excipients are those in which theinventive liposome active agent is sufficiently stable to allow fortherapeutic use, for example polyethylene glycols, certain fats, andesters or mixtures of these substances. Ointments, pastes, cream, andgels can also contain suitable excipients in which the liposome activeagent is stable. The composition also can be formulated for injection(e.g., intravenously, interstitially, intratumorally, etc) by theinclusion of one or more excipients (e.g., buffered saline) suitable forinjection.

The active agent or its pharmaceutical preparations can be administeredintravenously, subcutaneously, locally, orally (as emulsion or liquid orcapsule), parenterally, intraperitoneally and/or rectally or by directinjection into tumors or sites in need of treatment by such methods asare known or developed. Pharmaceutically active guggulphospholipids andtheir analogues and other bioactive molecules of the present inventioncan be administered topically, e.g., as a cream, skin ointment, dry skinsoftener, moisturizer, etc.

Where the composition includes one or more active agents (e.g., amixture of active agents), the invention provides for the use of thecomposition to prepare a medicament for the treatment of a disease. Inthis sense, the invention also provides a method for treating a human oranimal disease. In accordance with the inventive method, the inventivecomposition containing one or a mixture of active agents is exposed to(administered to) a human or animal patient in need of such treatment.In this manner, the active agent(s) are delivered to the patient. Thepreferred animal in the present invention is a mammal.

The method can be used to administer one or more active agents. It isthought to be general for active agents that are stable in the presenceof surfactants. Hydrophilic active agents are suitable and can beincluded in the interior of the liposome such that the liposome bilayercreates a diffusion barrier preventing it from randomly diffusingthroughout the body. Hydrophobic active agents are thought to beparticularly well suited for use in the present method because they notonly benefit by exhibiting reduced toxicity but they tend to be wellsolubilized in the lipid bilayer of liposome.

In certain embodiments wherein the active agent is polynucleotide, themethods of the present invention find application in the transfection ofa cell or cells with the polynucleotide. The methods of the embodimentcan be employed to transfect cells in vitro, or to deliver therapeuticor diagnostic polynucleotides to cells in vivo. For example, methods ofthese embodiments can be used to deliver genes to cells in culture or topatients in connection with gene therapy regimens. For therapeuticapplications, such embodiments of the invention thus provide methods ofgene therapy comprising, e.g., administering a pharmaceuticalcomposition comprising one or more nucleic acids to a patient in need oftreatment, wherein the composition comprises cationic lipid (forexample, formula XII). In some embodiments, the polynucleotide comprisesan expression construct encoding a gene, such that the gene is expressedwithin the cell after transfection in accordance with the inventivemethod.

A lipid composition of the present invention can be used to facilitatethe intercellular delivery of oligonucleotides such as DNA, mRNA,antisense oligonucleotides or siRNA, and sequences coding fortherapeutically active polypeptides, Thus catonic lipid-mediateddelivery of DNA, mRNA, antisense oligonucleotides, siRNA or proteins inaccordance with embodiments of the invention can provide therapy forvarious diseases, including but not limited to genetic diseases.

In some embodiments, the invention also provides a kit for transfectionof polynucleotides into cells. The kit includes, e.g., cationic lipid(for example, Compound XII) and can also include (but need not include)a desired polynucleotide for transfection, The kit can also include thereagents for facilitating transfection, such as buffers, culture medium,etc.

Examples of therapeutic polynucleotides include antisenseoligonucleotides such as antisense RNA or DNA sequences, smallinterfering RNA (siRNA), microRNA (miRNA), ribozymes, and aptamers. Insome embodiments. therapeutic polynucleotides target desired nucleicacid or amino acid sequences within cells, such as genes or geneproducts associated with a disease state (e.g., oncogenes or viralgenes). A preferred therapeutic polynucleotide for targeting desiredgenes is a 10 to 40-mer antisense polynucleotide or 10-40-mer siRNAsequences, preferably between 15 to 25-mer sequences. Whereoligonucleotides are included in the composition, they may contain oneor more modifications, e.g., phosphothioate linkages or 2′O-methylribose, e.g. to alter characteristics such as nuclease resistance,hybridization behavior, etc. In some preferred embodiments, apolynucleotide of the present invention comprises two phosphothioatelinkages. In certain embodiments comprising two phosphothioate linkages,a polynucleotide composition of the present invention may contain onephosphothioate linkage at each terminal end, however the linkages can bepresent anywhere from one end to the other end (e.g. in between theends) of an oligonucleotide. A polynucleotide can be single-stranded ordouble-stranded.

Diseases in which the compositions of the present invention find use fortreatment will depend on the selection of active agents, such asdescribed herein. In preferred embodiments, at least one active agentincorporated into the composition is an anticancer agent (e.g., achemotherapeutic agent) for use in treating cancer. Chemotherapeuticagents are well suited for such use. Liposome formulations or complexesincluding micelles, vesicles and emulsion containing chemotherapeuticagents may be injected directly into the tumor tissue for delivery ofthe chemotherapeutic agent directly to cancer cells. In someembodiments, e.g., after resection of a tumor, the liposome formulationcan be implanted directly into the resulting cavity, and/or may beapplied to the remaining tissue as a coating. In cases in which theliposome formulation or complex is administered after surgery, it ispossible to utilize liposome having larger diameters of about 1 micronsince they do not have to pass through the vasculature. Compositionsaccording to the present invention find use in any type of cancer, andparticularly in cancer in a mammal. Examples include, but are notlimited to, cancers of the head, neck, brain, blood, (e.g., leukemia,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia,lymphoma, myeloma), breast, lung, pancreas, bone, spleen, bladder,prostate, testes, colon, kidney, ovary and skin (e.g. Kaposi's sarcoma),bone marrow, liver, stomach, tongue, mouth and larynx. Activecompound-lipid complexes of the present invention also find use inreducing the tendency of cancer cells to develop a resistance to othertherapeutic agents such as anti-cancer agents, chemotherapy andradiation. Thus, other therapeutic agents can be advantageously employedwith the present invention in the formation of an active combination orby separate administration (e.g., other therapeutic agents may be usedbefore, after, or administered at the same time as the compositions ofthe invention).

The compositions of the present invention can be employed to treatinfections caused by numerous fungi and parasites, including but notlimited to, Acremonium sp., Aspergillus fumigatus, Aspergilluspneumonia, Blastomyces dermatitidis, Candida albicans, Candidaguillermondi, Candida Coccidioides immitis, Cryptococcus neoformans,Fusarium sp., Histoplasma capsulatum, Mucor mucedo, Rhodotorula sp.,Sporothrix schenckii, Acanthamoeba polyphaga, Entomophthora sp.,Histoplasma capsulatumm Leishmania brasiliensis, Rhizopus sp.,Rhodotorula sp., Torulopsis glabrata, Paracoccidioides brasiliensis.Additional fungal pathogens include Trichosporon, Muco, Alternaria,Bipolaris, Curvularia, etc

The compositions of present invention also find use in the treatment ofVisceral Leishmaniasis also called as Kala-azar and infections caused byLeishmania donovani complex, Ld. donovani, L.d infantum, L.d archihaldi,L.d chagasi, Phlehotomus sp. and Lutzomya logipalpis.

The compositions of present invention can also be employed to treatviral infections, such as those caused, by human immunodeficiency virus(HIV), herpes simplex viruses (HSV-1 and HSV2), hepatitis C virus (HCV),human papilloma virus (HPV) and cyotomegalovirus (CMV).

In some embodiments, the inventive active compound-lipidcomplex (forexample, a tacrolimus-lipid complex) is employed to treat rejectionreactions caused by organ transplantations and can be administered organor tissue transplantation, e.g., in a mammal. In this regard, theinvention provides a methods and compositions for preventing organ ortissue rejection comprising administering to a subject (e.g. a patienthaving an organ or tissue transplantation) a composition comprising acomplex of active compound lipid-complex and lipid(s) in an amountsufficient to prevent an organ or tissue rejection within the subject.

Yet other diseases for treatment using liposome formulations orcomplexes of the invention include (but are not limited to) preventionor treatment of abnormal cell growth, proliferation in inflammation,cholesterol lowering and weight loss, skin diseases, memory loss,Alzheimer disease, neoplastic and cardiovascular diseases,anti-hyperglycemic, cognition enhancer, skin ointment, and HIV.

Embodiments of the invention also are directed to methods of deliveringpharmaceutically active guggulphospholipids and their analogues asactive agents (or in combination with or without other active agents asmixture) to cells. The methods can be carried out by preparing liposomeor complexes including micelles, vesicles and emulsion that includeactive agents and pharmaceutically active guggulphospholipids, theiranalogues and/or other bioactive molecules of the present invention. Theliposome or complex is then delivered to a cell or cells, which can bein vitro or in vivo, as desired. In vivo administration can be achievedas described herein or as otherwise known to those of ordinary skill.For in vitro use, delivery of the active agent(s) can be carried out byadding the composition (e.g., liposome or complex) to the cell culturemedium, for example.

Pharmaceutical compositions of the present invention can be administeredby any route, including but not limited to administration intravenously,subcutaneously, orally or parenterally, intraperitoneally, topically,dermally, rectally, and vaginally.

The use of terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “including”, “having”, and “containing”are to be construed as open-ended terms meaning “including but notlimited to”) unless otherwise noted. The use of any and all examples, orexemplary language (e.g., “such as”) provided herein, is intended merelyto better illuminate the invention and does not pose a limitation on thescope of the invention unless otherwise claimed. No language in thespecifications should be constructed as indicating any non-claimedelement as essential to the practice of the invention.

Preferred embodiments of this invention are described, including thebest mode known to the inventors for carrying out the invention.Variations of those preferred embodiments can become apparent to thoseof ordinary skilled artisans to employ such variations as appropriate,and the inventors intend for the inventions to be practiced otherwisethan specifically described herein. Accordingly, this invention includesall modifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

All references, including publications, patent applications, and patentcited herein, including those in the list below and otherwise cited inthis specification, are hereby incorporated by reference in theirentireties, to the same extent as if each reference was individually andspecifically indicated to be incorporated by reference and were setforth in the entirely herein.

The following examples further illustrate the invention but, of course,should not be construed as in any way as limiting its scope.

EXAMPLE 1 Synthesis of E-Guggulsterol, Z-Guggulsterol Or A Mixture of E-and Z-Guggulsterol

This Example demonstrates a fast and convenient methods for thesynthesis of E-Guggulsterol and Z-Guggulsterol or a mixture of E- andZ-Guggulsterol (II A) from commercially available E-Guggulsterone andZ-Guggulsterone or mixture of E- and Z-Guggulsterone (I), Compound (IIA)can be synthesized via a synthetic route outlined in FIG. 3.

Guggulsterone (I) (5.0 g, 16.02 mmol) was dissolved in anhydroustetrahydrofuran (TI-IF) (50 ml) and added dropwise to a slurry oflithium aluminum hydride (Li AlH₄) (1.0 g, 26.35 mmol) in anhydrous THF(100 ml) over a period of 30 minutes, maintaining the temperaturebetween 20° C. to 25° C. After the mixture was stirred for 3 hours, theprogress of reaction was checked by TLC (hexane:ethyl

Acetate; 6: 4, v/v). After completion of reaction, the excess of LiAH₄was destroyed by adding ice-cold water in small portion. The organiclayer was washed with ice-cold water, dried over anhydrous sulfate,filtered, and evaporated in vacuo to yield 4, 17(20)-Pregnadiene-3β,16α-diol of the formula (2) (4.73 g).

The compound of formula (2) was dissolved in anhydrous methylenechloride (60 ml) and activated manganese(IV)oxide(MnO₂)(10.0 g; 115.02mmole) was added and stirred at room temperature for 1.0 hour and thenheated to 40° C. with stirring for 30 minutes, followed by stirringadditional 8.0 hours at room temperature. Progress of reaction waschecked by TLC (methylene chloride:acetone; 9:1,v/v) . After thereaction was completed, the excessive manganese oxide was filteredthrough celite pad under vacuum. The solvent was removed in vacuo togive the crude product as yellow viscous oil(3.66 g). The crude productwas purified over silica gel column by eluting first with methylenechloride followed by 2% acetone in methylene chloride. The fractionscontaining the pure product were pooled and concentrated in vacuo. Thesolid obtained was recrystallized in. methylene chloride/hexane (1:10,20 ml) to give a white solid of the title compound (II A) (1.67 g; Yield33.2%),

EXAMPLE 2 Preparation of Guggulsteryl Sulfate (VII)

This example demonstrates a method for preparing Guggulsteryl sulfate(VII) from E-guggulsterol and Z-guggulsterol or mixture of E andZ-guggulsterols (II A).The compound VII can be synthesized via thesynthetic route outlined in FIG. 4. To a solution of anhydrous pyridine(5 ml) and anhydrous methylene chloride (5 ml) under nitrogen,chlorosulfonic acid (0.5 ml) was added and stirred in an ice-water bathfor 15 minutes. Guggulsterols (IIA) (0.108 g, 0.343 mmol) was dissolvedin anhydrous methylene chloride (5 ml) and added to the solution ofpyridine-chlorosulfonate with stirring. The reaction mixture was stirredvigorously for 15 minutes in an ice-water bath. The ice-water bath wasthen removed and the reaction mixture was allowed to warm to roomtemperature. The reaction flask was then refluxed for 3 hours. Thesolvents were removed in vacuo to give a white solid, which wasconverted to sodium salt by adding 0.1 NaOH followed by drying widerhigh vacuum. The purity of the product was checked by TLC (methylenechloride:acetone; 9:1v/v). The crude solids were triturated with diethylether (25 ml×2) to remove impurities and then precipitated in methanoland filtered to provide the title compound (VII) (0.060 g; yield41.95%).

EXAMPLE 3 Preparation of Guggulmyristate

This example demonstrates a method for preparing Guggulmyristate, aspecific example of compound III, from E- guggulsterol andZ-gugguisterol or a mixture of E- and Z- guggulsterols (II A). CompoundIII can be synthesized via the synthetic route outlined in FIG. 5. To astirred solution of guggulsterols (IIA)(0.109 g, 0.348 mmol) inanhydrous methylene chloride(15 ml) was addedN,N′-dicyclohexylcarbodiimide(DCC) (1.58 g; 7.65 mmol), followed bymyristic acid (C14:0) (0.1873 g; 0.820 mmol) and catalytic amount of4-dimethylaminopyridine (DMAP) (0.05 g; 0.409 mmol). The reactionmixture was stirred overnight at room temperature. The progress ofreaction was monitored by TLC (hexane:ethyl acetate, 6:4, v/v). Aftercompletion of reaction, the resulting white precipitate of DCU wasfiltered and washed with methylene chloride. The solvent was removedusing a rotary evaporator to yield White solids (2.01 g). The crudeproduct was purified over silica gel column (40 g) by eluting first withhexane (200 ml) and then with hexane: ethyl acetate (90:10; v/v). Thefractions containing pure product were pooled and filtered through a 0.2μm acrodisc. The solvents were removed in vacuo and dried under highvacuum to give the title compound III (0.16 g; yield 87.6%).

EXAMPLE 4 Preparation of Guggulphosphatidic Acid (VI)

This example demonstrates a method for preparing Guggulphosphatidic acid(VI) from E-guggulsterol and Z-guggulsterol or a mixture of E andZ-guggulsterols (IIA). Compound VI can be synthesized via the syntheticroute outlined in FIG. 6. Guggulsterol (IIA) (0.150 g; 0.477mmol) wasdissolved in anhydrous pyridine (10 ml) under nitrogen, and phosphorusoxychloride [POC₃] (2 ml) was added drop wise and reaction mixture wasstirred at room temperature for 30 minutes, after which the reactionmixture was refluxed for 2 hours. The reaction mixture was then cooledto room temperature and pyridine was removed using a rotary evaporator.The residue was dissolved in chloroform (25 ml) and washed with water(50 ml×3), dried over anhydrous sodium sulfate, and filtered. Ammoniumhydroxide (2 ml) was added to the filtrate and the solvent was removedin vacuo. The crude product was precipitated in methylenechloride:acetone(1:10) at −20° C., filtered and dried under high vacuumto give the title compound VI (53mg ; yield 26.9%).

EXAMPLE 5 Preparation of Guggulphospholipid (V)

This example demonstrates a method for preparing Guggulphospholipid (V)from E-guggulsterol and Z-guggulsterol or mixture of E andZ-guggulsterols (IIA). Compound V can be synthesized via the syntheticroute outlined in FIG. 7. To a solution of Guggulsterols IIA (0.100 g;0.318 mmol) in anhydrous methylene chloride (5 ml) at 0° C.;N,N-diisopropylethylamine (DIPEA) (1.0 m1; 5.71 mmol) andN,N-diisopropylmethylphosphonamidic chloride (1.0 ml; 5.18 mmol) weresequentially added drop wise while maintaining the temperature between0° C.-5° C. The reaction mixture was stirred for 6 hours at 0° C. andthe progress of reaction was monitored by TLC (methylenechloride:acetone; 9:1). After complete consumption of Guggulsterols IIA,the ice-bath was removed and the mixture was gradually allowed to attainroom temperature. Tetrazole solution (0.45M in acetonitrile) (1.0 ml)was added followed by 1,2-dimyristoylglycerol (0.16 g; 0.312 mmol), andthe reaction mixture was stirred at room temperature for 24 hours. Thereaction mixture was then cooled to −40° C. tent-butylhydroperoxide (1ml) was added, and the mixture was stirred for 1 hour. The reactionmixture was gradually allowed to attain room temperature and stirringwas continued for additional 1 hour at room temperature. The contentswere diluted with methylene chloride (50 ml) and the organic layer waswashed successively with ice-cold 5% HCl (10 ml×2), aq. sodiumbicarbonate (50 ml×2), water (50 ml) and brine (50 ml), dried overanhydrous sodium sulfate and concentrated. The resulting intermediate 3was dried under high vacuum to give yellow viscous oil (1.16 g). Theresulting oil was purified over silica gel column (15g) eluting with 1%methanol in methylene chloride to produce the intermediate 3 (0.52 g).

To a stirred solution of the intermediate 3 (0.50 g; 0.553 mmol) in2-butanone (20 ml) was added sodium iodide (0.250 g, 1.66 mmole), andthe reaction mixture was refluxed for 2.5 hours and cooled to 25° C. andthen at −20° C. overnight. The sodium salt of title compound V wasfiltered and washed with chilled acetone to give 0.22 g (76.12%) aswhite solid.

EXAMPLE 6 Synthesis of Guggulphospholipid (V)

This example demonstrates a second approach for the synthesis ofGuggulphospholipid V from E-guggulsterol and Z-guggulsterol or mixtureof E and Z-guggulsterols (HA). Another approach for the synthesis ofcompound V is outlined in FIG. 8. The1,2-dimyristoyl-sn-glycero-3-phosphatidic acid sodium salt (4) (200 mg)was dissolved in chloroform: methanol (8:2,v/v) and was converted to itsfree acid form by washing with ice-cold 0.1N HCl, followed by washingwith deionized water. The organic layer was dried over anhydrous sodiumsulfate and concentrated. The resulting1,2-dimyristoyl-sn-glycero-3-phosphatidic acid, 5 (DMPA) (190 mg, 0.320mmol) was dried under high vacuum over P₂O₅ overnight before use.

To a stirred solution of guggulsterols (HA) (100 mg; 0.318 mmole) inanhydrous methylene chloride (15 ml) N,N-dicyclohexylcarbodiimide (DCC)(1.5 g; 7.6576 mmol) was added and stirred at room temperature for 5minutes. Compound 5 (DMPA) dissolved in anhydrous methylene chloride (5ml) was added to reaction mixture, followed by a catalytic amount of4-dimethylaminopyridine (DMAP) (25mg). The reaction mixture was stirredovernight at room temperature. The progress of reaction was monitored byTLC (methylene chloride:acetone; 9:1, v/v). After completion ofreaction, the resulting white precipitate of DCU was filtered and washedwith methylene chloride. The solvent was removed in vacuo to yieldviscous oily material (1.19 g). The crude product was purified oversilica gel column (20 g) packed in CHCl₃: MeORNH₄OH (75:20:5, v/v), andthe column was eluted in the same solvent system. The fractionscontaining pure product were pooled and filtered through a 0.2 μmacrodisc. The solvent was removed using a rotary evaporator and driedunder high vacuum to give the ammonia salt of the title compound V (115mg; yield 60.8%).

EXAMPLE 7 Preparation of Guggulphosphocholine

This example demonstrates a method for preparing Guggulphosphocholine(VIII) from E-guggulsterol and Z-guggulsterol or a mixture of E andZ-guggulsterols (IIA). Compound VIII can be synthesized via thesynthetic route outlined in FIG. 9. To a solution of guggulsterols IIA(0.150 g; 0.477 mmol) in anhydrous methylene chloride (10 ml) at 0° C.;N,N diisopropylethylamine (DIPEA) (100 μl) and 2-cyanoethyldiisopropylchlorophosphoramidite (120 μl) were sequentiallyadded at 0° C. The reaction mixture was stirred for 3 hours at 0° C. Theprogress of reaction was monitored by TLC (methylene chloride:acetone;9:1). After complete consumption of guggulsterols IIA, the ice-bath wasremoved and the mixture was gradually allowed to attain roomtemperature. Tetrazole solution (0.45M in acetonitrile) (5.0 ml) wasadded, followed by choline chloride (0.075 g; 0.537 mmol), and thereaction mixture was stirred at room temperature for 24 hours. Thereaction mixture was then cooled to −20° C. and tert-butylhydroperoxide(100 μl) was added and stirred for 1 hour at this temperature andgradually allowed to attain room temperature and continued stirring foradditional 1 hour at room temperature. The contents were diluted withmethylene chloride (50 ml) and the organic layer was washed successivelywith ice-cold 5% HCl (10 ml×2), aq. sodium bicarbonate (50 ml×2), water(50 ml) and brine (50 ml), dried over anhydrous sodium sulfate, andconcentrated. The resulting intermediate 6 was dried under high vacuumto give a semisolid. (1.02 g), A solution of the above intermediate 6 inacetonitrile (10 ml) was mixed with triethylamine (Et₃N)(1 ml) andstirred at room temperature for 24 hours. The contents were concentratedand purified by flash column chromatography with CHCl₃: MeOH: NH₄OH(75:20:5, v/v) as eluent to produce the ammonia salt of title compoundVIII as a white solid (0.11 g, 43.2%).

EXAMPLE 8 Particles Comprising Tacrolimus, Guggulsterol, and SoyLecithin

Tacrolimus (20 mg), Guggulsterol (8 mg), and Soy lecithin (792 mg) weremixed together in water (15 mL) and homogenized for 10 minutes. Thesuspension was sonicated at 45° C. for 10 minutes and then passesthrough high pressure homogenizer. The resulting suspension was thenmixed with 7.5% sucrose solution (5 mL) and lyophilized. The particlesize was determined using Nicomp particle sizer 380. The mean particlediameter amounted to less than 200 nm.

Particle Size Mean/Distributions (Volume Weighting) Mean Volume  67.2 nmWeighting Diameter 99% Distribution 200.7 nm 90% Distribution 115.4 nm80% Distribution  91.5 nm 75% Distribution  83.8 nm 50% Distribution 58.7 nm 25% Distribution  41.2 nm

EXAMPLE 9 Particles Comprising Paclitaxel, Guggulsterol, and Soylecithin

Paclitaxel (40 mg), Guggulsterol (14.73 mg), and Soy lecithin (1.58 g)were mixed together in water (15 mL) and homogenized for 10 minutes. Thesuspension was sonicated at 45° C. for 10 minutes and then passesthrough high pressure homogenizer. The resulting suspension was thenmixed with 7.5% sucrose solution (5 mL) and lyophilized. The particlesize was determined using Nicomp particle sizer 380. The mean particlediameter amounted to less than 200 nm.

Particle Size Mean/Distributions (Volume Weighting) Mean Volume 106.6 nmWeighting Diameter 99% Distribution  62.0 nm 90% Distribution  90.9 nm80% Distribution 133.8 nm 75% Distribution 189.6 nm 50% Distribution346.2 nm 25% Distribution 147.2 nm

REFERENCES

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What is claimed is:
 1. A guggul-based lipid molecule having a generalstructure selected from the group consisting of structures III-XII.

wherein: i) in structures III and. IV, R₁ is a saturated or unsaturatedacyl or alkyl groups having between 1 and 34 carbon atoms; ii) instructures III and IV, R₁ and R₂ is a saturated alkyl or alkyloxy groupoptionally substituted with amino or substituted amino group. ii) instructure IV, R₁ is a sugar; iii) in structure V, R₁ and R₂ are the sameor different and at least one of the R₁ or R₂ is a saturated orunsaturated acyl group or alkyl group having between 1 and 34 carbonatoms; iv) in structures IV-X, X is hydrogen, methyl, ammonium, sodium,potassium, calcium, barium ion or any non-toxic ion; v) in structure VI,X and Y are the same or different and are hydrogen, 1(i methyl,hydrogen, ammonium, sodium, potassium, calcium, barium ion or anynon-toxic ion; vi) in structure VIII, R₃, R₄ and R₅ are the same ordifferent and are hydrogen or methyl group; X is hydrogen or methylgroup. vii) in structure X, PEG (polyethylene glycol) is a long chain,linear or branched synthetic polymer composed of ethylene oxide units,HO(CH₂CH₂O)_(n)CH₂CH₂OCH₃, where n is between about 1 and about 1000;viii) in structure XI, drug is an active agent; and ix) in structureXII, R₆, R₇, and R₈ are the same or different, and are selected from thegroup consisting of hydrogen, methyl, alkyl, substituted alkyl, alkyoxy,substituted alkyloxy groups, and are optionally hydroxylated, aminated,or polyaminated such that the composition has an overall positivecharge; and wherein: x) structures III-XII are in the form selected fromthe group consisting o E-isomer, Z-isomer, or a mixture of E- andZ-isomers; xi) structures III-XII are optically pure, or one or more ofstructures III-XII are mixtures of optical isomers; xii) structure VIIIis cationic when X is a methyl group; and xiii) structure XII iscationic,
 2. A method of preparing a guggul derivative of a formulaselected from the group consisting of formula III-XII, comprisingreacting a guggulsterol having structure IIA

to form a composition comprising a guggul derivative of a formulaselected from the group consisting of formula III-XII.
 3. A method ofpreparing a guggul derivative of a formula selected from the groupconsisting of formula III-XII, comprising reacting a guggulsteronehaving structure I

to form a composition comprising a guggul derivative of a formulaselected from the group consisting of formula III-XII.
 4. The method ofclaim 3, wherein said guggulsterone I is an E-isomer or Z-isomer or amixture of E and Z isomers.
 5. The method of claim 2, wherein saidguggulsterol IIA is an E-isomer or Z-isomer, or a mixture of E- andZ-isomer.
 6. The method of claim 2, wherein said guggulsterol isoptically pure, and wherein said guggulsterol is a mixture of opticalisomers.
 7. A method of preparing guggulsterol IIA, comprising treatingguggulsterone I in one of more steps to produce preparing guggulsterolIIA.
 8. A method of preparing a compound of formula IV, V, VI, VIII, IXor X of claim 1, 2, or 3, wherein at least one step comprises the use ofa phosphoramidite reagent or a phosphorylating agent.
 9. The method ofclaim 8, wherein said phosphoramidite reagent is selected from the groupconsisting of NN-diisopropylmethylphosphoramidic chloride,(benzyloxy)(N,N-diisopropylamino)chlorophosphine, benzyloxybis(diisopropylamino) phosphine,2-cyanoethyl-N,N,N,N-tetraisopropylphosphoramidite,(2-cyanoethyl)(N,N-diisopropylamino)chlorophosphine, difluorenyldiisopropylphosphoramidite, methyl-N,N,N,Ntetraisopropylphosphorodiamidite, dimethylN,N-diisopropylphosphoramidite, dibenzyl diisopropylphosphoramidite,di-tert-butyl-N,N-diisopropylphosphoramidite,2-(diphenylmethylsilyl)ethyl-N,N,N,N-tetraisopropylphosphoramidite,(N-trifluoroacetyl amino) butyl and (N-trifluoroacetylamino)pentyl-N,N,N,N-tetraisopropylphosphoramidites.
 10. The method of claim8, wherein said phosphorylating agent is selected from the groupconsisting of 2-bromoethyldichlorophosphate, tri methylsilylethyldichlorophosphate, methyl dichlorophosphate,2-chloro-2-oxo-1,3,2-dioxaphospholane, 2-chlorophenyl dichlorophosphate,and phosphorus oxychloride.
 11. A method of producing a composition ofclaim 1, wherein compound XI is prepared by conjugating guggulsterolwith a drug directly or through a linker.
 12. The method of claimwherein compound XI is prepared by conjugating guggulsterol through alinker, wherein the linker is an alkyl group optionally substituted witha functional group selected from the groups consisting of
 13. A methodcomprising complexing a guggul-based lipid molecule of claim 1 into a.composition comprising a liposome or lipid complex.
 14. The method ofclaim 13, wherein said complex is selected from a group consisting ofmicelles, vesicles, and emulsions, wherein if said composition comprisesa plurality of micelles, said micelles are in the form of monomeric,dimeric, polymeric or mixed micelles.
 15. A method of retaining drug ina liposome or complex comprising providing one or more guggulderivatives of claim 1 or preparing one or more guggul derivatives by amethod of claims 2-11, and complexing said one or more guggulderivatives and a drug in a composition comprising a liposome orcomplex.
 16. The methods of claims 13-15, wherein said compositionfurther comprises at least one of a phospholipid, a pegylatedphospholipid, polyethylene glycol (PEG), a fatty acid, a sterol, cholicacid, or α-tocopherol.
 17. The method of claim 16, wherein saidcomposition comprises at least one phospholipid, wherein saidphopholipid comprises a phosphatidylcholine, a phosphatidylethanolamine,a phosphatidylglycerol, a phosphatidylserine, a phosphatidylinositol, ora phosphatidic acid.
 18. The method of claim 16, wherein saidcomposition comprises at least one polyethylene glycol (PEG), whereinsaid polyethylene glycol (PEG) has an average molecular weight of200-20,000.
 19. The method of claim 16, wherein said compositioncomprises at least one fatty acid, wherein said fatty acids is selectedfrom a group consisting of fatty acids having chain length of C₄-C₃₄.20. The method of claim 19, wherein said fatty acid is saturated orunsaturated, and wherein said fatty acid is in acidic form, or in saltform.
 21. The method of claim 16, wherein said composition comprises atleast one pegylated phospholipid selected from the group consisting of apegylated derivative of disteroylhosphatidylglycerol,dimyristoylhosphatidylglycerol, or dioleoylphosphtidylglycerol.
 22. Themethod of claim 16, wherein said cholic acid is sodium deoxycholate. 23.The method of claim 16, wherein said composition further comprises asterol selected from a group consisting of cholesterol, derivatives ofcholesterol, cholesterol sulfate, cholesterol succinate, cholesterolhemisuccinate, cholesterol oleate, cortisol, corticosterone,hydrocortisone, cholesterol-PEG, coprostanol, cholestanol, cholestane,β-sitosterol, lanosterol, campesterol, lathosterol, stigmasterol,stigmastanol, calciferol, guggulsterols, derivatives of guggulsterols,and mixtures thereof.
 24. The method of claims 15, wherein said drugcomprises a therapeutically active agent.
 25. The method of claim 24,wherein said therapeutically active agent comprises an anticancer drug,an antiviral drug, antibacterial drug an antifungal drug,anti-inflammation drug, cholesterol lowering drug, and wherein at leastone of said therapeutically agent is to treat cardiovascular disease, totreat neoplasia, to improve memory lose, to treat skin infection or skindisease, to treat hyperglycemia, or to enhance cognitive function.
 26. Amethod wherein at least one cationic guggul derivative of formulas VIIIand XII of claim 1 is used to facilitate intercellular delivery of apolynucleotide.
 27. A method of transfecting a cell with apolynucleotide, comprising (a) preparing a composition comprising acationic guggul derivative of claim 1 and said polynucleotide, and (b)contacting said cell with said composition whereby said polynucleotideis taken up by the cell.
 28. The method of claim 26 or 27, wherein saidpolynucleotide is selected from a group consisting of an oligonucleotidecomprising DNA, RNA or DNA and RNA, antisense oligonucleotides, smallinterfering RNA (siRNA), microRNA (miRNA), aptamers, and ribozymes, andwherein said polynucleotide is a single-stranded or double-stranded. 29.The method of any of claims 14-28, wherein said composition comprisesunilamellar vesicles, multilamellar vesicles, or mixtures thereof. 30.The method of any of claims 13-29 wherein said liposome or complex hasan overall charge selected from a negative charge, a positive charge, ora neutral charge.
 31. The method of any of claims 13-30, wherein theliposome or complex has a mean diameter of about 20 micron to less than1 micron.
 32. The method of any of claims 13-30, wherein the liposome orcomplex has a mean diameter of about 500 nm to less than 100 nm.
 33. Themethod of any of claims 13-32, wherein said composition is inlyophilized form.
 34. The method claim 33, wherein said composition inlyophilized form comprises a cryoprotectant, wherein said cryoprotectantcomprises one or more sugars are selected from a group consisting oftrehalose, maltose, lactose, sucrose, and dextran.
 35. The method of anyof claims 13-33, wherein said liposome or complex is in a powder form orin a solution form, in a suspension form, in an emulsion form, andwherein said liposome or complex is in a gel form, in micelle form, orin a paste form.
 36. The method of any of claims 13-35, wherein saidcomposition is a tablet or is tilled in a capsule.
 37. A method oftreating a cell, comprising preparing composition according to any ofthe claim 1, or 13-36, and exposing said cell to said composition, 38.The method of claim 37, wherein said cell is in vivo in a subject.
 39. Amethod of treating a human or animal disease, comprising administering atherapeutically effective amount of the composition prepared accordingto a method of claim 1, 13-36 and exposing the composition to a human oranimal in need thereof such that the composition is delivered to thehuman or animal patient.
 40. A kit of transfecting cells, said kitcomprising a cationic guggul derivative of claim 1 and one or moreelements selected from the group consisting of a polynucleotide,instructions for formulating the cationic guggul derivative andpolynucleotide into a preparation, instructions for transfecting cellsusing the cationic guggul derivative, reagents for facilitatingtransfection, containers for storing the cationic guggul derivative,containers for storing the polynucleotide, containers for storing thereagents, containers for storing a preparation including the cationicguggul derivative and polynucleotide, or containers for preparing thepreparation, and materials to facilitate transfection.
 41. A compositionproduced according to a method of any of claim 2-25 or 27-36.